CN101878228A - Human monoclonal nicotine specific antibodies - Google Patents

Human monoclonal nicotine specific antibodies Download PDF

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CN101878228A
CN101878228A CN2008801181882A CN200880118188A CN101878228A CN 101878228 A CN101878228 A CN 101878228A CN 2008801181882 A CN2008801181882 A CN 2008801181882A CN 200880118188 A CN200880118188 A CN 200880118188A CN 101878228 A CN101878228 A CN 101878228A
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seq
peptide
monoclonal antibody
nicotine
lcvr
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M·巴克曼
M·鲍尔
R·贝尔利
P·莫勒
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Cytos Biotechnology AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/34—Tobacco-abuse
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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Abstract

The invention relates to recombinantly produced human monoclonalantibodies which are specifically binding nicotine and to nucleic acids encoding the same.The invention further relates to the use of such antibodies in the treatment of nicotine addiction.

Description

Human monoclonal nicotine specific antibodies
Technical field
The present invention relates to recombinate the specificity that produces in conjunction with Nicotine and can stop the human monoclonal antibodies of Nicotine from blood transfer to brain.The invention further relates to the application of these antibody in passive immunization, the preferably application in people's passive immunization, and relate to the application of these antibody in treatment people's nicotine addiction.
Background technology
People .1986 such as Bjercke (J Immunol Methods, Vol.90 (2) pp.203-13) disclose the stereospecificity monoclonal antibody of anti-(S)-(-)-Nicotine.The monoclonal anti of people .1986 such as Bjercke-Nicotine antibody produces by hybridoma technology, therefore derives from mouse cell.People .1986 such as Bjercke have further described these monoclonal antibodies and have developed based on the application in the nicotine analysis of ELISA.
People .2001 (J.Org.Chem.Vol.66pp.4115-4121) such as Isomura have described haptenic preparation, and this haptens is used to produce the specific antibody of naturally occurring (S)-Nicotine with high-optical-purity, (S)-and (R)-nornicotine and metabolite (S)-cotinine.Authors report the preliminary data of anti-Nicotine antibody (comprising monoclonal antibody).The monoclonal antibody of people .2001 such as Isomura obtains from mouse, and shows 1.6x10 -7M to 3.4x10 -7The Nicotine of M is in conjunction with the Kd value.
People .2005 such as Keyler (Drug Metab Dispos, Vol.33 (7) pp.1056-61) have described and have used the passive immunization of anti-Nicotine monoclonal antibody to rat.Nicotine reduced to the distribution of brain after the author especially found passive immunization.The monoclonal antibody of people .2005 such as Keyler also produces by hybridoma technology.
People .2006 such as Pentel (J Pharmacol Exp Ther, Vol.317 (2) pp.660-6) especially studied the passive immunization of monoclonal antibody Nic311 to rat, this antibody is described by people .2005 such as Kepler, studied especially thus use this antibody passive immunization to Different Effects acute and that the chronic nicotine brain distributes.Pointed out in the prior art and used nicotine specific antibody that people's passive immunization is come in handy in the treatment of nicotine addiction, and may in the smoking cessation process, be favourable.But about Nic311, people .2006 such as Pentel further draw to draw a conclusion: therapeutic monoclonal antibodies need carry out the immunogenicity that humanization reduces himself just can be used for clinical application.
The known anti-Nicotine monoclonal antibody of prior art comes from inhuman source, and therefore, because their strong immunogenicities in human body and the security consideration that causes thus, they are disabled on treating.
Summary of the invention
The invention provides the human monoclonal antibodies of specificity in conjunction with Nicotine, wherein preferably described monoclonal antibody is a fully human antibodies.Therefore typical case and preferably, monoclonal antibody disclosed herein does not have immunogenicity in human body is applicable to human passive immunization.In addition, be surprised to find, monoclonal antibody specificity of the present invention in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine.With (S)-(-)-Nicotine is the same, (R)-(+)-steric isomer of Nicotine also is present in the tobacco plant.In addition, the combustion processes when it also can be by smoking forms, and have the activity to central nervous system similar in nature to (S)-(-)-Nicotine (people such as Hukkanen J.., 2005, Pharmacol.Rev.57,79-115).
Monoclonal antibody of the present invention is cloned according to disclosed method among the PCT/EP2007/061570: (1) selects human experimenter's nicotine specific B cell; (2) VH and the VL district of these cells are cloned in the Alphavirus library; (3) on mammalian cell, carry out the cell surface display of scFv antibody; (4) cell of nicotine specific scFv antibody is showed in selection; (5) clone the VH and the VL district of these scFv antibody; (6) with the monoclonal antibody of different formal representation specificitys, for example as the scFv-Fc-fusion rotein or as the human IgG2, preferably as complete human IgG2 in conjunction with Nicotine.By using this method, obtained 7 independently antibody clonings, when they are expressed as scFv-Fc fusion rotein or IgG2, with the Kd value of nmole or low nmole scope in conjunction with Nicotine.
Therefore, on the one hand, the present invention relates to the monoclonal antibody of specificity in conjunction with Nicotine, wherein said monoclonal antibody is a human monoclonal antibodies, and wherein preferably described human monoclonal antibodies is complete human monoclonal antibodies.
With these cloning and sequencings, and identify the people CDR that nicotine specific is provided.Be surprised to find several identical CDR (seeing Table 1) in these antibody with various combination.
The SEQ ID NO of the CDR sequence of table 1. Nicotine-human antibodies specific
Further, the present invention relates to the monoclonal antibody of specificity in conjunction with Nicotine, wherein preferably described monoclonal antibody is a human monoclonal antibodies, and wherein said monoclonal antibody comprises at least one variable region of heavy chain (HCVR), wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 comprises or preferably is made up of the peptide of any among the SEQ ID NO:1,11 and 17, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 comprise or preferably are made up of the peptide of any among the SEQ ID NO:2,12 and 18, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; And/or (c) HC CDR3, wherein said HC CDR3 comprises or preferably is made up of the peptide of any among the SEQ IDNO:3,4,13 and 19, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR.
Further, the present invention relates to the monoclonal antibody of a specific specificity in conjunction with Nicotine, wherein preferably described monoclonal antibody is a kind of human monoclonal antibodies, wherein said monoclonal antibody comprises at least one variable region of light chain (LCVR), wherein said LCVR comprises: (a) LC CDR1, wherein said LC CDR1 comprise or preferably are made up of the peptide of any among the SEQ ID NO:5,6,7,14 and 20; And wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 comprise or preferably are made up of the peptide of any among the SEQ ID NO:8,15 and 21, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; And/or (c) LC CDR3, wherein said LC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:9,10,16 and 22, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
Further, the present invention relates to the monoclonal antibody of a specific specificity in conjunction with Nicotine, wherein preferably described monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HCCDR1, wherein said HC CDR1 comprises or preferably is made up of the peptide of any among the SEQ ID NO:1,11 and 17, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 comprise or preferably are made up of the peptide of any among the SEQ ID NO:2,12 and 18, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; And/or (c) HC CDR3, wherein said HC CDR3 comprises or preferably is made up of the peptide of any among the SEQ IDNO:3,4,13 and 19, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And/or preferably, wherein said monoclonal antibody comprises at least one LCVR, and wherein said LCVR comprises: (a) LCCDR1, wherein said LC CDR1 comprise or preferably are made up of the peptide of any among the SEQ ID NO:5,6,7,14 and 20; And wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LCCDR2 comprise or preferably are made up of the peptide of any among the SEQ ID NO:8,15 and 21, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; And/or (c) LC CDR3, wherein said LC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:9,10,16 and 22, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
Typical case and preferably, described monoclonal antibody is that reorganization produces.Monoclonal antibody of the present invention can be with any naturally occurring or synthetic formal representation.In a preferred embodiment, described monoclonal antibody is IgG, preferably IgG2.
Further, the present invention relates to the HCVR of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, and wherein said HCVR comprises: (a) HC CDR1, and wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of any in SEQ ID NO:3 and 4.
Further, the present invention relates to the LCVR of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, and wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of any among the SEQ ID NO:5,6 and 7; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of any in SEQ ID NO:9 and 10.
Further, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-100nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-100nM.In a preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-70nM, 1-40nM preferably, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-70nM, 1-50nM preferably, 1-40nM further preferably, 1-20nM further preferably again, 1-10nM more further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.In a highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-70nM, 1-40nM preferably, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-20nM, and 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.Preferably, described monoclonal antibody is a kind of human monoclonal antibodies, wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HCCDR1 comprises or preferably is made up of the peptide of any among the SEQ ID NO:1,11 and 17, most preferably form, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR by the peptide of SEQ ID NO:1; (b) HC CDR2, wherein said HC CDR2 comprises or preferably is made up of the peptide of any among the SEQ ID NO:2,12 and 18, most preferably form, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR by the peptide of SEQ ID NO:2; And/or (c) HCCDR3, wherein said HC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:3,4,13 and 19, most preferably form, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR by the peptide of SEQ ID NO:3; And/or preferably, wherein said monoclonal antibody comprises at least one LCVR, and wherein said LCVR comprises: (a) LC CDR1, wherein said LC CDR1 comprise or preferably are made up of the peptide of any among the SEQ ID NO:5,6,7,14 and 20; Most preferably form, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR by the peptide of SEQ ID NO:5; (b) LC CDR2, wherein said LCCDR2 comprises or preferably is made up of the peptide of any among the SEQ ID NO:8,15 and 21, most preferably form, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR by the peptide of SEQ ID NO:8; And/or (c) LC CDR3, wherein said LC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:9,10,16 and 22, most preferably form, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR by the peptide of SEQ ID NO:9.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-10nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-10nM, wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:9, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR, and wherein preferably the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ IDNO:26.
Further, the present invention relates to the to encode nucleic acid molecule of variable region of the present invention, monoclonal antibody of the present invention or its single chain.
Further, the present invention relates to comprise the expression vector of at least a nucleic acid molecule of the present invention.Further, the present invention relates to comprise the host cell of at least a nucleic acid molecule of the present invention or at least a expression vector of the present invention.
Monoclonal antibody of the present invention can be used for passive immunization, is preferably used for people's passive immunization, and is used for the treatment of nicotine addiction.Therefore, further, the present invention relates to comprise the pharmaceutical composition of at least a monoclonal antibody of the present invention.
Further, the present invention relates to a kind of passive immunization method, described method comprises the pharmaceutical composition of the present invention of using the monoclonal antibody of the present invention or the significant quantity of significant quantity to the experimenter.
Further, the present invention relates to a kind of method for the treatment of nicotine addiction, described method comprises pharmaceutical composition of the present invention from significant quantity to the experimenter that use the monoclonal antibody of the present invention or the significant quantity of.
Further, the present invention relates to be used for passive immunization, be preferably used for the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention of people's passive immunization.
Further, the present invention relates to be used for the treatment of nicotine addiction, be preferably used for treating the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention of people's nicotine addiction.
Further, the present invention relates to monoclonal antibody of the present invention is used for the medicine of passive immunization (preferably at Nicotine) in preparation purposes.
Further, the present invention relates to purposes in monoclonal antibody of the present invention is used for the treatment of (preferably people) nicotine addiction in preparation the medicine.
Another aspect of the present invention is the application of antibody of the present invention in the method for quantitative and/or qualitative detection Nicotine, preferably detects blood sample, most preferably detects by ELISA.
Should be appreciated that all embodiments disclosed herein and technical characterictic all relate to all aspects of the present invention and any possible combination.
Description of drawings
The specificity of Fig. 1 .IgG2-F018.In the presence of unlabelled (-)-Nicotine, (-)-cotinine or vagusstoff that concentration increases gradually, detect combining of tritium-labeled (-)-Nicotine (56nM) and IgG2-F018 (50nM) by equilibrium dialysis.The mean value and the standard deviation of two independent experiments have been provided.
Detailed Description Of The Invention
" antibody ": term used herein " antibody " refers to a kind of molecule, a kind of protein preferably, and it can a kind of antigen of specific binding, typical case and preferably, by determining in conjunction with the epi-position of described antigen or antigen bunch, or in conjunction with haptens. Preferably, term antibody refers to comprise antigen or the haptens binding molecule at least one variable district, and wherein preferably described molecule comprises at least one HCVR and/or at least one LCVR. Further preferably, term antibody refers to comprise at least one, preferably just in time two antigen is in conjunction with antigen or the haptens binding molecule in site, and wherein said antigen is formed by a HCVR and a LCVR in conjunction with in the site each. In addition, term antibody also refers to complete antibody, the antibody of preferred IgG, IgA, IgE, IgM or IgD classification, the more preferably antibody of IgG classification, the most preferably antibody of IgG1, IgG2, IgG3 and IgG4 classification, and antigen binding fragment section. In a preferred embodiment, described complete antibody comprises κ or lambda light chain. Term " antibody " also refers to antigen or haptens in conjunction with antibody fragment, optimization protein hydrolysis fragment and the similar thing of recombinating, most preferably Fab, Fab ' and F (ab ') 2 and Fv. Term antibody further comprise comprise at least one, the protein in preferred two variable districts, wherein further preferably described protein comprises just what a HCVR and just what a LCVR. In a preferred embodiment, term antibody refers to single-chain antibody, preferred scFv. Therefore, preferred antibody is single-chain antibody, the Fv (sdFv) that preferred scFv, disulfide bond connect and comprise light chain variable district (LCVR) or the fragment of weight chain variable district (HCVR). In the context of the present invention, term " antibody " preferably refers to recombinant antibodies, comprises the restructuring albumen that is comprised of a polypeptide, wherein said polypeptide comprise at least one, preferred what a variable district just. In the context of the present invention, recombinant antibodies can further comprise function element, such as joint district, signal peptide or hydrophobic leading sequence, tags detected and/or purification tag (for example Fc).
" Fv ": term Fv refers to can be in conjunction with the proteolytic fragments of the minimum of antigen or haptenic antibody, and the similar thing of the restructuring of described fragment.
" single-chain antibody ": the antibody that single-chain antibody is comprised of a polypeptide. Preferred single-chain antibody by comprise at least one, preferably just the polypeptide of what a VR forms, wherein preferably described VR is HCVR. Preferred single-chain antibody by comprise at least one, preferably just what a HCVR and at least one, preferably just the polypeptide of what a LCVR forms. Preferred single-chain antibody comprises just what a HCVR and just what a LCVR again. Single-chain antibody most preferably is scFv, wherein said scFv by one just comprise what a HCVR and just the polypeptide of what a LCVR form, wherein said HCVR and described LCVR are connected to each other by the joint district, wherein preferably described joint district by at least 15, preferably 15-20 amino acid form (people such as Bird. (1988) Science, 242 (4877): 423-426). Further preferred single-chain antibody is scFv, wherein said scFv is encoded by a code area, wherein said code area 5 ' to 3 ' direction, comprise with following order: (1) is by the light chain variable district (LCVR) that forms from the light chain framework (LC FR) 1 of κ or lambda light chain, complementary determining area (LC CDR) 1, LC FR2, LC CDR 2, LC FR3, LC CDR3 and LCFR4; (2) flexible joint (L); (3) the weight chain variable district (HCVR) that is formed by framework (HCFR) 1, complementary determining area (HC CDR) 1, HC FR2, HC CDR2, HC FR3, HCCDR3 and HC FR4. Perhaps, single-chain antibody is scFv, wherein said scFv is encoded by a code area, wherein said code area 5 ' to 3 ' direction, comprise with following order: the weight chain variable district (HCVR) that (1) is comprised of framework (HC FR) 1, complementary determining area (HC CDR) 1, HC FR2, HC CDR2, HC FR3, HC CDR3 and HC FR4; (2) flexible joint (L); (3) by the light chain variable district (LCVR) that forms from the light chain framework (LC FR) 1 of κ or lambda light chain, complementary determining area (LC CDR) 1, LC FR2, LC CDR2, LC FR3, LC DR3 and LC FR4.
" double-chain antibody (diabody) ": term " double-chain antibody " refers to comprise two polypeptide chains, the antibody of two identical polypeptide chains preferably, wherein every polypeptide chain comprises HCVR and LCVR, wherein said HCVR and described LCVR are connected to each other by the joint district, wherein preferably described joint district comprise maximum 10 amino acid (people such as Huston. (1988), PNAS85 (16): 587958-83; The people such as Holliger. (1993), PNAS 90 (14): 6444-6448, and Hollinger Hudson, 2005, Nature Biotechnology 23 (9): 1126-1136; The people such as Arndt. (2004) FEBS Letters 578 (3): 257-261). The preferred joint district of double-chain antibody comprises 0,1,2,3,4,5,6,7,8,9 or 10 amino acid.
" people's antibody ": term used herein " people's antibody " refers to basically have the antibody of the white amino acid sequence of people's immune globulin, preferred recombinant antibodies, or its fragment, and comprise the antibody that from people's immune globulin text of an annotated book storehouse, separates. In the context of the present invention, " people's antibody " can comprise the amino acid of comparing with the natural human antibody sequence exchange of limited quantity. These amino acid exchanges can be for example, to be caused by clone's process. But the number that these amino acid exchange in people's antibody of the present invention is minimumization preferably. Preferably, the sequence at least 85% of the amino acid sequence of people's antibody and natural human antibody, preferably 90%, more preferably 95%, even more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical. More preferably, the sequence at least 85% of the amino acid sequence of people's antibody and specific binding purpose antigen or haptenic natural human antibody, preferably 90%, more preferably 95%, even more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical. Most preferably, the sequence at least 85% of the natural human antibody of the amino acid sequence of people's antibody and specific binding nicotine, preferred (S)-(-)-nicotine and/or (R)-(+)-nicotine, preferably 90%, more preferably 95%, more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical.
Preferred recombinant human antibody has at most 20,15,10,9,8,7,6,5,4,3,2 or 1 amino acid different from natural human antibody, most preferably, eliminate the difference of the amino acid sequence of recombinant human antibody and natural human antibody by molecular cloning, therefore, most preferably, recombinant human antibody is identical with the amino acid sequence of natural human antibody. This kind antibody is also referred to as " fully human antibodies ". Embodiment 6 provides an illustrative embodiment that how obtains fully human antibodies from the people's antibody that is selected from people's antibody library. Typical case and preferably, fully human antibodies does not have immune originality in human body.
Preferred people's antibody comprises: (a) at least one, a preferred HCVR, (b) at least one, a preferred HCCR, (c) at least one, a preferred LCVR, and (d) at least one, a preferred LCCR, wherein said at least one HCVR and/or described at least one HCCR and/or described at least one LCVR and/or described at least one LCCR and corresponding natural human zone at least 85%, preferably 90%, more preferably 95%, more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical.
Determine that the constant district of immune globulin white (comprising that people's immune globulin is white) exists with multiple type of the same race, that is, the amino acid sequence in described constant district may have difference to a certain degree between the individuality of colony. Studied the type of the same race in the white constant district of people's immune globulin, the technical staff obtains sequence informations from various sources easily very deeply, comprise Immuno GeneticsInformation System (http://imgt.cines.fr/). Should be appreciated that for purpose of the present invention the type different of the same race in the constant district that a kind of immune globulin is white can exchange. For example, γ 2 heavy chains of monoclonal antibody of the present invention can comprise the type of the same race of any existence of people γ 2HCCR.
" monoclonal antibody ": term used herein " monoclonal antibody " refers to include only the antibody colony of an antibody type (that is the antibody that, has same acid sequence).
" constant district (CR) ": term " constant district " refers to the constant district of light chain (LCCR) or the constant district of heavy chain (HCCR) of antibody. Typical case and preferably, described CR comprise 1-4 the immunoglobulin domains take the ring structure of disulfide bond stabilisation as feature. Preferred CR is the white CR of immune globulin, the white CR of people's immune globulin preferably, and wherein further preferably, described immune globulin is white, and preferably described people's immune globulin is white, is selected from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM and IgD. CR very preferably is the people CR that comprises or be comprised of the amino acid sequence that can obtain from public database, and described database comprises, for example, Immunogenetic Information System (http://imgt.cines.fr/)。
The constant district of light chain (LCCR): LCCR, more particularly κ LCCR or λ LCCR, the natural κ of the natural antibody of general proxy or the C-end portion of lambda light chain. LCCR generally comprises about 110 amino acid, represents an immunoglobulin domains.
The constant district of heavy chain (HCCR): the constant district of heavy chain comprise the antibody heavy chain about 3/4ths or more, and it is terminal to be positioned at its C-. HCCR generally comprises 3 or 4 immunoglobulin domains. Preferred HCCR is selected from γ HCCR, α HCCR, ε HCCR, our HCCR and δ HCCR. It is most preferred that γ HCCR, wherein preferably described γ HCCR is selected from γ 1HCCR, γ 2HCCR, γ 3HCCR and γ 4HCCR, and wherein most preferably described γ HCCR is γ 2HCCR.
" variable district (VR) " but refer to variable district or the variable domain of antibody, more specifically refer to weight chain variable district (HCVR) or light chain variable district (LCVR). Typical case and preferably, VR comprises an immunoglobulin domains. Preferred VR is the white VR of immune globulin, the preferred white VR of people's immune globulin, and wherein further preferably, described immune globulin is white, and preferably described people's immune globulin is white, is selected from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM and IgD. The VR of various species is known in the art. Preferred VR is people VR, and the framework of wherein said people VR shows that the framework with any known person VR sequence has at least 80%, preferably at least 85%, more preferably 90%, more preferably at least 95%, and at least 99% sequence homogeneity most preferably. Preferred VR is people VR, and the framework of wherein said people VR shows the framework with anyone the VR sequence that can obtain from public database, most preferably with can from ImmunogeneticsInformation System (http://imgt.cines.fr/) anyone VR sequence of obtaining, have at least 80%, preferably at least 85%, more preferably 90%, more preferably at least 95%, at least 99% sequence homogeneity most preferably.
Each VR comprises so-called complementary determining area (CDR), and complementary determining area determines the binding characteristic of antibody, and is embedded in the so-called framework. Typical case and preferably, VR comprises 3 CDR, is preferably CDR1, CDR2 and CDR3, they are embedded in the framework (FR 1-4). Therefore, in a preferred embodiment, VR holds the C-end to comprise in the following order following element: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-.
Usually, VR comprises polypeptide or preferably is comprised of polypeptide, and wherein said polypeptide is for example product of D and J constant gene segment C (HCVR) or J constant gene segment C (LCDR) combination of the member of V-constant gene segment C family and other constant gene segment C.
" light chain variable district (LCVR) ": the light chain variable district is by the nucleic acid molecule encoding of resetting, and is κ LCVR or λ LCVR. People κ LCVR comprises a polypeptide, and wherein said polypeptide is the member's of the people κ V-constant gene segment C 1-7 of family product. In the context of the present invention, preferred κ LCVR is people κ LCVR, preferably by a kind of people κ LCVR of dna encoding, this DNA can use PCT/EP2007/061570 as disclosed any oligonucleotides of SEQ ID NO:49-52 and the PCT/EP2007/061570 combination of primers as disclosed any oligonucleotides of SEQ ID NO:53-56, further preferably, use the embodiment 3 described PCR conditions of PCT/EP2007/061570, increase from human B cell.
People λ LCVR comprises a polypeptide, and wherein said polypeptide is the member's of the people λ V-constant gene segment C 1-11 of family product. In the context of the present invention, preferred λ LCVR is people λ LCVR, preferably by a kind of people λ LCVR of dna encoding, this DNA can use any the combination of primers among the SEQ ID NO:66-68 of any and PCT/EP2007/061570 among the SEQ ID NO:57-65 of PCT/EP2007/061570, further preferably, use the embodiment 3 described PCR conditions of PCT/EP2007/061570, increase from human B cell.
Typical case and preferably, LCVR comprises three LC CDR, preferably LC CDR1, LC CDR2 and LC CDR3, they are embedded in the light chain framework (LC FR 1-4). Therefore, in a preferred embodiment, LCVR holds the C-end to comprise in the following order following element from N-: LC FR1-LC CDR1-LC FR2-LC CDR2-LC FR3-LC CDR3-LCFR4.
" weight chain variable district (HCVR) ": the weight chain variable district is by the nucleic acid molecule encoding of resetting. People HCVR comprises a polypeptide, and wherein said polypeptide is member's the product of the 1-7 of family of people λ V-constant gene segment C. In the context of the present invention, preferred HCVR is people HCVR, preferably by a kind of people HCVR of dna encoding, this DNA can use the combination of primers of the SEQ ID NO:48 of any and PCT/EP2007/061570 among the SEQ ID NO:42-47 of PCT/EP2007/061570, further preferably, use the embodiment 3 described PCR conditions of PCT/EP2007/061570, increase from human B cell.
Typical case and preferably, HCVR comprises three HC CDR, preferably HC CDR1, HC CDR2 and HC CDR3, they are embedded in the heavy chain framework (HC FR 1-4). Therefore, in a preferred embodiment, HCVR holds the C-end to comprise in the following order following element from N-: HC FR1-HC CDR1-HC FR2-HC CDR2-HC FR3-HC CDR3-HCFR4.
" CDR ": the complementary determining area (CDR) 1,2 and 3 of the HCVR of antibody and LCVR can be identified respectively by means commonly known in the art. For the application, CDR and FR border are such as definition as described in the people .2003 (Developmental and Comparative Immunology Vol.27pp.55-77) such as the people .1999 such as Scavinger (Exp Clin Immunogenet., Vol.16pp.234-240) or Lefranc.
" antigen ": term used herein " antigen " refers to by the molecule of antibody combination. Antigen is replied identification by immune system and/or body fluid immunity, and may have one or more epi-positions, and preferably B cell epitope, or antigen determines bunch.
" haptens ": term " haptens " typically refers to a kind of chemical compound, wherein said compound itself can not be in animal induce immune response, but wherein said compound when be combined with carrier, can be in animal during preferably with the carrier protein combination induce immune response. In the context of the present invention, " haptens " refers to nicotine.
" nicotine ": term used herein " nicotine " preferably refers to (S)-(-)-nicotine or (R)-(+) nicotine or its any mixture. Most preferably, nicotine refers to (S)-(-)-nicotine.
" specific binding ": the specificity of antibody is relevant with antibody specific binding antigen or haptenic ability. The specificity of this kind interaction (affinity) is used in conjunction with constant and is characterized between antibody and the antigen/haptens, and perhaps on the contrary, (Kd) characterizes with the constant that dissociates. Should be appreciated that antibody depends on antibody and antigen/haptenic structure to antigen/haptenic apparent affinity in the multivalence interaction, and depend on the practical measurement condition. Because affinity, antibody may be significantly higher than the unit price interaction to antigen/haptenic apparent affinity during multivalence interacted. Therefore, preferably under the condition that is conducive to the unit price interaction, determine affinity. Kd can determine with methods known in the art. The Kd of antibody and antigen/haptenic given combination preferably determines by ELISA, the wherein continuous dilution liquid contact of the antibody purification (preferred univalent antibody, for example scFv or Fab fragment) of the fixing antigen/haptens of constant amount and concentration known. Then with observe the half maximum in conjunction with the time antibody concentration determine Kd. In a preferred embodiment, preferably under embodiment 7 described conditions, determine Kd by the balance dialysis. Perhaps, the Kd that antibody and antigen/haptenic unit price interacts analyzes conduct in conjunction with speed (k by Biacoreon) and the speed (k that dissociatesoff) ratio determine. Compare the antibody that lower Kd value representation specificity is higher and antigen/haptenic combination with higher numerical value. In the application's context, when the constant that dissociates (Kd) (preferably measure as mentioned above, further preferably measure in unit price interacts) mostly is 1mM (＜=10 most-3M), preferably mostly be most 1 μ M (＜=10-6M), most preferably mostly be most 1nM (＜=10-9M) time, antibody is considered to " specific binding antigen/haptens ". It is most preferred that and with the Kd that is lower than 100nM (" low receive molar range ") in conjunction with antigen/haptenic antibody, wherein further preferably, in unit price interacts, to measure Kd. Further preferred antibody can be with 1-1000nM, more preferably with 5-800nM, more preferably with 5-90nM, most preferably with the Kd of 5-50nM in conjunction with antigen/haptens, wherein further preferably in unit price interacts, measure Kd.
" equate ": in the application's context, when the value of two dissociation constants (Kd) of monoclonal antibody and material differs maximum 10 times, preferred maximum 9 times, more preferably maximum 8 times, more preferably maximum 7 times, more preferably maximum 6 times, more preferably maximum 5 times, more preferably maximum 4 times, more preferably maximum 3 times, more preferably maximum 2 times, most preferably maximum 1.5 times the time, described value is considered to " equating ".In a highly preferred embodiment, when two Kd values differed less than 5 times, they were considered to equate.
" significant quantity ": the significant quantity of monoclonal antibody of the present invention or pharmaceutical composition of the present invention typically refers at the dosage of necessity and is enough to the amount of the treatment result that obtains to wish in the time period, wherein preferably described result prevents, reduces or slow down Nicotine entering in (preferably people) brain, and/or, final smoking cessation.Therapeutic for the people is disposed, and " significant quantity " generally is meant 1mg to 1000mg, preferably 10mg to 500mg, more preferably 10mg to 300mg, more preferably 50mg to 200mg, the most preferably amount of about described monoclonal antibody of 100mg.
" label ": term tag, preferably purifying or detection label are meant the polypeptide section that can be connected on second polypeptide with purifying or detection that second polypeptide is provided or the site that second polypeptide is connected with substrate is provided.In principle, any peptide or the protein that can obtain its antibody or other specific-binding agent can be used as the avidity label.Label comprises hemagglutinin label, myc label, polyhistidyl label, albumin A, glutathione s-transferase, Glu-Glu avidity label, P material, FLAG peptide, Streptavidin binding peptide or other antigenic epitopes or in conjunction with territory (great majority from US06686168).
Disclose among the PCT/EP2007/061570 a kind ofly be used to identify, separation and clone-specific binding purposes antigen or haptenic scFv based on the library screening method.Particularly, described method can identify, separate and clone people scFv and produce fully human antibodies subsequently comprises Fab fragment and entire I gG.By using described technology, identified and cloned the human monoclonal antibodies of specificity in conjunction with Nicotine.
On the one hand, the invention provides the monoclonal antibody of specificity in conjunction with Nicotine, wherein said monoclonal antibody is a human monoclonal antibodies, wherein preferably described Nicotine is selected from (S)-(-)-Nicotine and (R)-(+)-Nicotine, and wherein most preferably described Nicotine is (S)-(-)-Nicotine; And wherein further preferably, described human monoclonal antibodies is complete human monoclonal antibodies.
In a preferred embodiment, described monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine.
In one embodiment, described monoclonal antibody is in conjunction with Nicotine, preferably in conjunction with (S)-(-)-Nicotine, its dissociation constant Kd is 1-1000nM, more preferably 1-800nM, more preferably 1-100nM, most preferably 5-90nM, wherein further preferably, use the described monoclonal antibody measuring Kd of IgG2 form.In a further preferred embodiment, Kd measures in unit price interacts, and most preferably measures in substantially as embodiment 7 described analyses.
In a further preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-100nM; And the bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-100nM.
In a preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 0.1-100nM, 0.1-70nM preferably, 0.1-50nM further preferably, 0.1-40nM further preferably again, 0.1-20nM further preferably again, 0.1-10nM further preferably, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-100nM, 0.1-70nM preferably, 0.1-50nM further preferably, 0.1-40nM further preferably again, 0.1-20nM further preferably again, and 0.1-10nM further preferably again, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.In another highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 0.1-40nM, 0.1-10nM further preferably, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-50nM, 0.1-20nM preferably, 0.1-10nM further preferably, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.In another highly preferred embodiment again, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 0.1-10nM, 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-10nM, and 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form.
In a further preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-1000nM, 1-800nM preferably, 1-100nM more preferably, 1-70nM further preferably again, 1-50nM further preferably again, 1-40nM further preferably again, 1-20nM further preferably again, 1-10nM still more preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-1000nM, 1-800nM preferably, 1-100nM more preferably, 1-70nM further preferably again, 1-50nM further preferably again, 1-40nM further preferably again, 1-20nM further preferably again, 1-10nM still more preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.In a highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-100nM, 1-70nM preferably, 1-40nM further preferably again, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-100nM, 1-70nM preferably, 1-50nM further preferably again, 1-40nM further preferably again, 1-20nM further preferably again, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.In another highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-70nM, 1-40nM preferably, 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-50nM, 1-20nM preferably, and 1-10nM further preferably wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.
In a further preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 100nM, be preferably lower than 70nM, further preferably be lower than 50nM, further preferably be lower than 40nM again, further preferably be lower than 20nM again, and further preferably be lower than 10nM again, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form; And the described dissociation constant Kd of described bonded of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 100nM, be preferably lower than 70nM, further preferably be lower than 50nM, further preferably be lower than 40nM again, further preferably be lower than 20nM again, and further preferably be lower than 10nM again, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form.In a highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 70nM, be preferably lower than 40nM, further preferably be lower than 10nM, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 50nM, be preferably lower than 20nM, further preferably be lower than 10nM, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form.In another highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 20, is preferably lower than 10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form.
In a further preferred embodiment, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, wherein said monoclonal antibody equates with the described bonded dissociation constant Kd of described (R)-(+)-Nicotine with the described bonded dissociation constant Kd and the described monoclonal antibody of described (S)-(-)-Nicotine, wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form.
In a further preferred embodiment, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine be described monoclonal antibody and described (R)-(+)-Nicotine described bonded dissociation constant Kd 2-5 doubly, 2 times of preferably approximatelies, most preferably 2 times, wherein preferably use the described monoclonal antibody measuring Kd of IgG2 form.
In a further preferred embodiment, described monoclonal antibody Nicotine the entering in (preferably people) brain that can prevent, reduce or slow down.In a further preferred embodiment, described monoclonal antibody can reduce Nicotine entering in brain, when under substantially as embodiment 8 described conditions, measuring, preferably be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.Most preferably, described monoclonal antibody can reduce Nicotine entering in brain, when measuring under substantially as embodiment 8 described conditions, preferably is reduced by at least 60%.
In a further preferred embodiment, when being applied to the experimenter, preferably be applied to man-hour, described monoclonal antibody Nicotine the entering in described experimenter (preferably described people) brain that can prevent, reduce or slow down, wherein preferably described monoclonal antibody be with 1mg to 1000mg, preferably 10mg to 500mg, 10mg to 300mg more preferably, 50mg to 200mg more preferably again, most preferably approximately the amount of 100mg is applied to described experimenter, preferably is applied to described people.
The specificity of antibody depends primarily on the CDR of the variable region of light chain (LCVR) of the aminoacid sequence of complementary determining region (CDR) in the variable region of heavy chain (HCVR) of described antibody and/or described antibody.The invention discloses the CDR (HC CDR) of the HCVR of monoclonal antibody and the CDR (LC CDR) of LCVR, wherein said monoclonal antibody can specificity in conjunction with Nicotine.
Therefore, in a preferred embodiment, described monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HCCDR1 comprises or preferably is made up of the peptide of any among the SEQ ID NO:1,11 and 17, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 comprise or preferably are made up of the peptide of any among the SEQ IDNO:2,12 and 18, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; And/or (c) HC CDR3, wherein said HC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:3,4,13 and 19, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR.
In a further preferred embodiment, described monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, wherein said LCCDR1 comprise or preferably are made up of the peptide of any among the SEQ ID NO:5,6,7,14 and 20; And wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 comprise or preferably are made up of the peptide of any among the SEQ ID NO:8,15 and 21, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; And/or (c) LCCDR3, wherein said LC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:9,10,16 and 22, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
Be surprised to find, some can have the identical HC CDR and/or the LC CDR (referring to table 1) of various combination with the Kd of nmole scope monoclonal antibody, particularly F018, F063, J004, J042 and the N049 in conjunction with Nicotine.In addition, be surprised to find, the heavy chain of antibody F018, F063, J004, J042 and N049 is very relevant each other with variable region of light chain, only in several amino acid position difference.
Therefore, in a highly preferred embodiment, (a) described HC CDR1 comprises or preferably is made up of the peptide of SEQ ID NO:1; (b) described HC CDR2 comprises or preferably is made up of the peptide of SEQ ID NO:2; And (c) described HC CDR3 comprises or preferably is made up of the peptide of any in SEQ ID NO:3 and 4, and wherein preferably (a) described LC CDR1 comprises or preferably is made up of the peptide of any among the SEQ ID NO:5,6 and 7; (b) described LCCDR2 comprises or preferably is made up of the peptide of SEQ ID NO:8; And (c) described LC CDR3 comprises or preferably is made up of the peptide of any in SEQ ID NO:9 and 10.
In a preferred embodiment, described HCVR comprises a framework, and wherein said framework is the product that member and the JH-constant gene segment C of the VH4 family of the member of the VH family of V-constant gene segment C, preferred V-constant gene segment C makes up.
In a highly preferred embodiment, (a) described HC CDR1 is made up of the peptide of SEQ IDNO:1; (b) described HC CDR2 is made up of the peptide of SEQ ID NO:2; And (c) described HC CDR3 is made up of the peptide of SEQ ID NO:3.
In a further highly preferred embodiment, (a) described HC CDR1 is made up of the peptide of SEQID NO:1; (b) described HC CDR2 is made up of the peptide of SEQ ID NO:2; And (c) described HC CDR3 is made up of the peptide of SEQ ID NO:4.
In one embodiment, the 7-117 position of described HCVR is made up of the peptide of any among the SEQ IDNO:24,28,33 and 39.In another embodiment, the 7-117 position of described HCVR is by any nucleic acid encoding among the SEQ ID NO:23,27,32,29 and 38.
Antibody of the present invention may and different with fully human antibodies at some amino acid position place of VR, wherein these positions generally be positioned near the N-end and/or C-end of variable region owing to clone artefact (cloning artifacts).Preferably, from antibody, remove these artefacts in order to obtain fully human antibodies.A kind of method that produces complete human monoclonal antibodies from the antibody that still comprises described clone's artefact has been described among the embodiment 6.A kind of similar method is applied to all HCVR and LCVR disclosed herein.For example, in order to obtain complete people HCVR, the 1-6 position of HCVR of the present invention is replaced with SEQ ID NO:50.Therefore, in a preferred embodiment, the 1-6 position of described HCVR is made up of SEQ ID NO:50.
In a further embodiment, described LC CDR1 comprises or preferably is made up of the peptide of any among the SEQ ID NO:5,6 and 7; (b) described LC CDR2 comprises or preferably is made up of the peptide of SEQ ID NO:8; And (c) described LC CDR3 comprises or preferably is made up of the peptide of any in SEQ ID NO:9 and 10.
In further embodiment, described LCVR comprises a framework, and wherein said framework is the product that member and the J λ constant gene segment C of V λ 1 family of the member of the V λ family of V-constant gene segment C, preferred V-constant gene segment C makes up.
In a preferred embodiment, described LC CDR1 is made up of the peptide of SEQ ID NO:5; (b) described LC CDR2 is made up of the peptide of SEQ ID NO:8; And (c) described LC CDR3 is made up of the peptide of SEQ ID NO:9.
In a further preferred embodiment, (a) described LC CDR1 is made up of the peptide of SEQ IDNO:6; (b) described LC CDR2 is made up of the peptide of SEQ ID NO:8; And (c) described LC CDR3 is made up of the peptide of SEQ ID NO:10.In a further preferred embodiment, (a) described LC CDR1 is made up of the peptide of SEQ ID NO:7; (b) described LC CDR2 is made up of the peptide of SEQ ID NO:8; And (c) described LC CDR3 is made up of the peptide of SEQ ID NO:10.In a further preferred embodiment, (a) described LC CDR1 is made up of the peptide of SEQ IDNO:5; (b) described LC CDR2 is made up of the peptide of SEQ ID NO:8; And (c) described LC CDR3 is made up of the peptide of SEQ ID NO:10.
In a highly preferred embodiment, the 5-107 position of described LCVR is made up of the peptide of any among the SEQID NO:26,31,35,37 and 41.In further highly preferred embodiment, the 5-107 position of described LCVR is by any nucleic acid encoding among the SEQ ID NO:25,30,34,36 and 40.In a further preferred embodiment, the 1-4 position of described LCVR is made up of SEQ ID NO:51.In a further preferred embodiment, the 108-110 position of described LCVR is made up of SEQ ID NO:53.
In a further highly preferred embodiment, (a) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26; (b) the 7-117 position of described HCVR is made up of the peptide of SEQ IDNO:28, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:31; (c) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:33, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:35; (d) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:28, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:37; Perhaps the 7-117 position of (e) described HCVR is made up of the peptide of SEQ ID NO:39, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:41.In the further highly preferred embodiment of another one, (a) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26.
A further preferred embodiment, described specificity comprises at least one HCVR and at least one LCVR in conjunction with the human monoclonal antibodies of Nicotine, the 7-117 position of wherein said HCVR and SEQ ID NO:24,28,33 and 39 7-117 position, preferably with the 7-117 position at least 80% of SEQID NO:24, preferably 85%, more preferably 90%, again more preferably 95%, even more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical; And the 5-107 position of wherein said LCVR and SEQ ID NO:26,31,35,37 and 41 5-107 position, preferably with the 5-107 position at least 80% of SEQ ID NO:26, preferably 85%, more preferably 90%, more preferably 95%, even more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical.
Whether a kind of peptide or amino acid sequence of polypeptide are determined with alternative aminoacid sequence 80%, 85%, 90%, 95%, 97% or 99% identical known computer program such as the Bestfit program routine can used at least.
In a further preferred embodiment, described specificity comprises at least one HCVR and at least one LCVR in conjunction with the human monoclonal antibodies of Nicotine, the 7-117 position of wherein said HCVR and SEQ ID NO:24,28,33 and 39 7-117 position, preferably with the 7-117 position at least 80% of SEQ IDNO:24, preferably 85%, more preferably 90%, more preferably 95%, even more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical; And the 5-107 position of wherein said LCVR and SEQ ID NO:26,31,35,37 and 41 5-107 position, preferably with the 5-107 position at least 80% of SEQ IDNO:26, preferably 85%, more preferably 90%, more preferably 95%, even more preferably at least 96%, more preferably 97%, more preferably 98%, more preferably 99%, most preferably 100% is identical, and wherein said HCVR comprises: (i) HC CDR1 who is made up of the peptide of SEQ ID NO:1; (ii) HC CDR2 who forms by the peptide of SEQ ID NO:2; (iii) HC CDR3 who forms by the peptide of SEQ ID NO:3, and wherein said LCVR comprises: (i) LCCDR1 who is made up of the peptide of SEQ ID NO:5; (ii) LC CDR2 who forms by the peptide of SEQ ID NO:8; (iii) LC CDR3 who forms by the peptide of SEQ ID NO:9.
In a further highly preferred embodiment, (a) the 7-117 position of described HCVR is by the nucleic acid encoding of SEQ ID NO:23, and the 5-107 position of wherein said LCVR is by the nucleic acid encoding of SEQ ID NO:25; (b) the 7-117 position of described HCVR is by the nucleic acid encoding of SEQID NO:27, and the 5-107 position of wherein said LCVR is by the nucleic acid encoding of SEQ IDNO:30; (c) the 7-117 position of described HCVR is by the nucleic acid encoding of SEQ ID NO:32, and the 5-107 position of wherein said LCVR is by the nucleic acid encoding of SEQ ID NO:34; (d) the 7-117 position of described HCVR is by the nucleic acid encoding of SEQ ID NO:29, and the 5-107 position of wherein said LCVR is by the nucleic acid encoding of SEQ ID NO:36; Perhaps the 7-117 position of (e) described HCVR is by the nucleic acid encoding of SEQ ID NO:38, and the 5-107 position of wherein said LCVR is by the nucleic acid encoding of SEQ ID NO:40.
The form that antibody of the present invention can be used as single-chain antibody produces.Therefore, in a further highly preferred embodiment, described monoclonal antibody comprises or preferably is made up of the peptide of any among the SEQ IDNO:60,62,64,66 and 68.In a further preferred embodiment, described monoclonal antibody is a kind of single-chain antibody, and wherein preferably described single-chain antibody comprises or preferably is made up of the peptide of any among the SEQ ID NO:60,62,64,66 and 68.In further highly preferred embodiment, described monoclonal antibody comprises or preferably is made up of a peptide, and wherein said peptide is by any nucleic acid encoding among the SEQ ID NO:59,61,63,65 and 67.
In a further preferred embodiment, described monoclonal antibody is expressed as IgG, preferably is expressed as IgG2.Therefore, in a further preferred embodiment, described monoclonal antibody comprises at least one γ 2 heavy chain, and wherein preferably described γ 2 heavy chains comprise or preferably are made up of the peptide of any among the SEQ ID NO:73,75,77 and 80.In a further preferred embodiment, described monoclonal antibody comprises at least one lambda light chain, and wherein preferably described lambda light chain comprises or preferably is made up of the peptide of any among the SEQ ID NO:74,76,78,79 and 81.
In a highly preferred embodiment, described monoclonal antibody comprises at least one γ 2 heavy chain and at least one lambda light chain, wherein (a) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ IDNO:73, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:74; (b) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:75, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:76; (c) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:77, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:78; (d) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:75, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:79; Perhaps (e) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:80, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:81.
In a further preferred embodiment, described monoclonal antibody comprises constant region of light chain (LCCR), and wherein preferably described LCCR is λ LCCR.
In another embodiment, (a) described HC CDR1 comprises or preferably is made up of the peptide of SEQID NO:11; (b) described HC CDR2 comprises or preferably is made up of the peptide of SEQ IDNO:12; And (c) described HC CDR3 comprises or preferably is made up of the peptide of SEQ IDNO:13, wherein preferably described HCVR comprises a framework, and wherein said framework is the member of the member of the VH family of V-constant gene segment C, preferred V-constant gene segment C VH4 family and the product of JH-constant gene segment C combination.
In a preferred embodiment, (a) described HC CDR1 is made up of the peptide of SEQ ID NO:11; (b) described HC CDR2 is made up of the peptide of SEQ ID NO:12; And (c) described HCCDR3 is made up of the peptide of SEQ ID NO:13.
In a further preferred embodiment, the 7-121 position of described HCVR is made up of the peptide of SEQ ID NO:43.In a further preferred embodiment, the 7-121 position of described HCVR is by the nucleic acid encoding of SEQ ID NO:42.And in a further preferred embodiment, the 1-6 position of described HCVR is made up of SEQ ID NO:50.
In a further preferred embodiment, (a) described LC CDR1 comprises or preferably is made up of the peptide of SEQ ID NO:14; (b) described LC CDR2 comprises or preferably is made up of the peptide of SEQ ID NO:15; And (c) wherein said LC CDR3 comprises or preferably is made up of the peptide of SEQ ID NO:16, wherein preferably described LCVR comprises a framework, the product of the member of the member of the V κ family that wherein further preferably described framework is the V-constant gene segment C, preferred V-constant gene segment C V κ 3 families and the combination of J kappa gene section.
In a further preferred embodiment, (a) described LC CDR1 is made up of the peptide of SEQ IDNO:14; (b) described LC CDR2 is made up of the peptide of SEQ ID NO:15; And (c) described LC CDR3 is made up of the peptide of SEQ ID NO:16.In a further preferred embodiment, the 5-104 position of described LCVR is made up of the peptide of SEQ ID NO:45.In a further preferred embodiment, the 5-104 position of described LCVR is coded by the nucleic acid of SEQ ID NO:44.And in a further preferred embodiment, the 1-4 position of described LCVR is made up of SEQ ID NO:52.And in a further preferred embodiment, the 105-107 position of described LCVR is made up of SEQ ID NO:54.
In a highly preferred embodiment, the 7-121 position of described HCVR is made up of the peptide of SEQID NO:43, and the 5-104 position of described LCVR is made up of the peptide of SEQ ID NO:45.In a highly preferred embodiment, the 7-121 position of described HCVR is by the nucleic acid encoding of SEQ ID NO:42, and the 5-104 position of described LCVR is by the nucleic acid encoding of SEQ IDNO:44.
In a further highly preferred embodiment, described monoclonal antibody comprises or preferably is made up of the peptide of SEQ ID NO:70.In a further highly preferred embodiment, described monoclonal antibody is a kind of single-chain antibody, and wherein said single-chain antibody comprises or preferably is made up of the peptide of SEQ ID NO:70.In a further highly preferred embodiment, described monoclonal antibody comprises or preferably is made up of a peptide, and wherein said peptide is by the nucleic acid encoding of SEQ ID NO:69.
In a further highly preferred embodiment, described monoclonal antibody comprises at least one γ 2 heavy chain, and wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:82.In a further highly preferred embodiment, described monoclonal antibody comprises at least one κ light chain, and wherein said κ light chain comprises or preferably is made up of the peptide of SEQ ID NO:83.In a further highly preferred embodiment, described monoclonal antibody comprises at least one γ 2 heavy chain and at least one κ light chain, wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQID NO:82, and wherein said κ light chain comprises or preferably is made up of the peptide of SEQ IDNO:83.
In a further preferred embodiment, described monoclonal antibody comprises constant region of light chain (LCCR), and wherein preferably described LCCR is κ LCCR.
In further embodiment, (a) described HC CDR1 comprises or preferably is made up of the peptide of SEQ ID NO:17; (b) described HC CDR2 comprises or preferably is made up of the peptide of SEQ IDNO:18; And (c) described HC CDR3 comprises or preferably is made up of the peptide of SEQ IDNO:19, wherein preferably described HCVR comprises a framework, and wherein said framework is the member of the member of the VH family of V-constant gene segment C, preferred V-constant gene segment C VH4 family and the product of JH-constant gene segment C combination.
In a preferred embodiment, (a) described HC CDR1 is made up of the peptide of SEQ ID NO:17; (b) described HC CDR2 is made up of the peptide of SEQ ID NO:18; And (c) described HCCDR3 is made up of the peptide of SEQ ID NO:19.In a highly preferred embodiment, the 7-121 position of described HCVR is made up of the peptide of SEQ ID NO:47.In a further highly preferred embodiment, the 7-121 position of described HCVR is by the nucleic acid encoding of SEQ ID NO:46.In a further preferred embodiment, the 1-6 position of described HCVR is made up of SEQ ID NO:50.
In further embodiment, (a) described LC CDR1 comprises or preferably is made up of the peptide of SEQ ID NO:20; (b) described LC CDR2 comprises or preferably is made up of the peptide of SEQ IDNO:21; And (c) wherein said LC CDR3 comprises or preferably is made up of the peptide of SEQ IDNO:22, wherein preferably described LCVR comprises a framework, and wherein said framework is the member of the member of the V λ family of V-constant gene segment C, preferred V-constant gene segment C V λ 1 family and the product of J λ constant gene segment C combination.
In a preferred embodiment, (a) described LC CDR1 is made up of the peptide of SEQ ID NO:20; (b) described LC CDR2 is made up of the peptide of SEQ ID NO:21; And (c) described LCCDR3 is made up of the peptide of SEQ ID NO:22.
In a further preferred embodiment, the 5-107 position of described LCVR is made up of the peptide of SEQ ID NO:49.In a further preferred embodiment, the 5-107 position of described LCVR is by the nucleic acid encoding of SEQ ID NO:48.And in a preferred embodiment, the 1-4 position of described LCVR is made up of SEQ ID NO:51.And in a preferred embodiment, the 108-110 position of described LCVR is made up of SEQ ID NO:53.
In a highly preferred embodiment, the 7-121 position of described HCVR is made up of the peptide of SEQID NO:47, and the 5-107 position of described LCVR is made up of the peptide of SEQ ID NO:49.
In a highly preferred embodiment, the 7-121 position of described HCVR is by the nucleic acid encoding of SEQID NO:46, and the 5-107 position of described LCVR is by the nucleic acid encoding of SEQ ID NO:48.
In a preferred embodiment, described monoclonal antibody comprises or preferably is made up of the peptide of SEQID NO:72.In a further preferred embodiment, described monoclonal antibody is a kind of single-chain antibody, and wherein said single-chain antibody comprises or preferably is made up of the peptide of SEQ ID NO:72.In a preferred embodiment, described monoclonal antibody comprises or preferably is made up of a peptide, and wherein said peptide is by the nucleic acid encoding of SEQ ID NO:71.
In a further preferred embodiment, described monoclonal antibody comprises at least one γ 2 heavy chain, and wherein preferably described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:84.
In a further preferred embodiment, described monoclonal antibody comprises at least one lambda light chain, and wherein said lambda light chain comprises or preferably is made up of the peptide of SEQ ID NO:85.
In a highly preferred embodiment, described monoclonal antibody comprises at least one γ 2 heavy chain and at least one lambda light chain, wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ IDNO:84, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:85.
In a further preferred embodiment, described monoclonal antibody comprises constant region of light chain (LCCR), and wherein preferably described LCCR is λ LCCR.
Monoclonal antibody of the present invention can be used as naturally occurring form or the reorganization of synthetic form produces.Following embodiment describes all aspects of the present invention and embodiment in detail.In one embodiment, described monoclonal antibody is a kind of recombinant antibodies.In a preferred embodiment, monoclonal antibody of the present invention is to be selected from following antibody: (a) single-chain antibody, preferably scFv; (b) Fab fragment; (c) F (ab ') 2 fragments; (d) scFv-Fc fusion rotein; (e) IgG1; (f) IgG2; (g) IgG3; (h) IgG4; (i) IgA; (j) IgE; (k) IgM; (1) IgD; (m) double-stranded antibody.
In a preferred embodiment, described monoclonal antibody comprise or preferably by what a HCVR just and/or just what a LCVR form.
In a further preferred embodiment, described monoclonal antibody comprises CH (HCCR), wherein preferably described HCCR is selected from (a) γ HCCR, (b) α HCCR, (c) ε HCCR, (d) our HCCR, (e) δ HCCR, wherein further preferably, described HCCR, preferably described γ HCCR, α HCCR, ε HCCR, our HCCR and/or δ HCCR are people HCCR.
In a further preferred embodiment, described HCCR is γ HCCR, and wherein preferably described γ HCCR is selected from (a) γ 1HCCR; (b) γ 2HCCR; (d) γ 3HCCR; (c) γ 4HCCR; Wherein preferably described γ HCCR is γ 2HCCR.
In a further preferred embodiment, described monoclonal antibody comprises constant region of light chain (LCCR), and wherein preferably described LCCR is selected from (a) λ LCCR; (b) κ LCCR, wherein further preferably described LCCR, most preferably described λ LCCR and/or described κ LCCR are people LCCR.
Preferably, monoclonal antibody of the present invention is IgG2, most preferably is complete human IgG2.Therefore, in a preferred embodiment, described monoclonal antibody comprises two, preferred two described γ 2 heavy chains just in time, and wherein further preferably, described two, preferably described just in time two described γ 2 heavy chains are identical.
In a further preferred embodiment, described monoclonal antibody comprises two, two light chains just in time preferably, and wherein preferably described light chain is selected from (a) lambda light chain; (b) κ light chain; Wherein further preferably, described two, preferably described just in time two described light chains are identical.
Preferably, described monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 comprises or preferably is made up of the peptide of any among the SEQID NO:1,11 and 17, most preferably form, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR by the peptide of SEQ ID NO:1; (b) HC CDR2, wherein said HC CDR2 comprises or preferably is made up of the peptide of any among the SEQ ID NO:2,12 and 18, most preferably form, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR by the peptide of SEQID NO:2; And/or (c) HC CDR3, wherein said HC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:3,4,13 and 19, most preferably form, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR by the peptide of SEQ ID NO:3; And/or preferably, wherein said monoclonal antibody comprises at least one LCVR, and wherein said LCVR comprises: (a) LCCDR1, wherein said LC CDR1 comprise or preferably are made up of the peptide of any among the SEQ ID NO:5,6,7,14 and 20; Most preferably form, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR by the peptide of SEQ ID NO:5; (b) LC CDR2, wherein said LC CDR2 comprises or preferably is made up of the peptide of any among the SEQ IDNO:8,15 and 21, most preferably form, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR by the peptide of SEQ ID NO:8; And/or (c) LC CDR3, wherein said LC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:9,10,16 and 22, most preferably form, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR by the peptide of SEQID NO:9.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody is in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HCCDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:9, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 0.1-10nM, preferably 1-10nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-10nM, and 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HCCDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3, and wherein preferably described HCCDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LCCDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:9, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 0.1-10nM, preferably 1-10nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-10nM, and 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises γ 2 heavy chains and a lambda light chain, wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:73, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:74.
In a further preferred embodiment, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine equals, preferably 2-5 doubly to, more preferably about 2 times to, most preferably 2 times combine with described (R)-(+)-the described of Nicotine to described monoclonal antibody, wherein preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:9, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
Preferably, described monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 comprises or preferably is made up of the peptide of any among the SEQID NO:1,11 and 17, most preferably form, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR by the peptide of SEQ ID NO:1; (b) HC CDR2, wherein said HC CDR2 comprises or preferably is made up of the peptide of any among the SEQ ID NO:2,12 and 18, most preferably form, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR by the peptide of SEQID NO:2; And/or (c) HC CDR3, wherein said HC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:3,4,13 and 19, most preferably form, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR by the peptide of SEQ ID NO:3; And/or preferably, wherein said monoclonal antibody comprises at least one LCVR, and wherein said LCVR comprises: (a) LCCDR1, wherein said LC CDR1 comprise or preferably are made up of the peptide of any among the SEQ ID NO:5,6,7,14 and 20; Most preferably form, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR by the peptide of SEQ ID NO:5; (b) LC CDR2, wherein said LC CDR2 comprises or preferably is made up of the peptide of any among the SEQ IDNO:8,15 and 21, most preferably form, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR by the peptide of SEQ ID NO:8; And/or (c) LC CDR3, wherein said LC CDR3 comprises or preferably is made up of the peptide of any among the SEQ ID NO:9,10,16 and 22, most preferably form, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR by the peptide of SEQID NO:9.
At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 10nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 10nM, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HCCDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:9, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody is in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HCCDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 10-100nM, preferably 25-75nM, most preferably 25-35nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-10nM, and 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HCCDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 10-100nM, preferably 25-75nM, most preferably 25-35nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-10nM, and 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; Described monoclonal antibody comprises γ 2 heavy chains and a lambda light chain, and wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ IDNO:75, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:76.
In a further preferred embodiment, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine equals, preferably 2-7 doubly to, more preferably about 5 times to, most preferably 5 times combine with described (R)-(+)-the described of Nicotine to described monoclonal antibody, wherein preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 40nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 10nM, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HCCDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody is in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HCCDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:7, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 10-100nM, preferably 25-75nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-20nM, and 10-15nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HCCDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HCCDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LCCDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:7, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 10-100nM, preferably 25-75nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-20nM, and 10-15nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises γ 2 heavy chains and a lambda light chain, wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:77, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:78.
In a further preferred embodiment, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine equals, preferably 2-7 doubly to, more preferably about 5 times to, most preferably 5 times combine with described (R)-(+)-the described of Nicotine to described monoclonal antibody, wherein preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:7, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 75nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 15nM, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HCCDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:7, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HCCDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 20-60nM, preferably 30-50nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-10nM, and 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HCCDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HCCDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LCCDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 20-60nM, preferably 30-50nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-10nM, and 1-10nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; Wherein said monoclonal antibody comprises γ 2 heavy chains and a lambda light chain, and wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:75, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:79.
In a further preferred embodiment, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine equals, preferably 2-7 doubly to, more preferably about 4 times to, most preferably 4 times combine with described (R)-(+)-the described of Nicotine to described monoclonal antibody, wherein preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 60nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 10nM, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HCCDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HCCDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HCCDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 20-80nM, preferably 30-75nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-15nM, and 1-12nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HCCDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HCCDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LCCDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
In a further embodiment very preferably, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 20-80nM, preferably 30-75nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 0.1-15nM, and 1-12nM preferably wherein preferably uses the described monoclonal antibody measuring Kd of IgG2 form; Wherein said monoclonal antibody comprises γ 2 heavy chains and a lambda light chain, and wherein said γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:80, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:81.
In a further preferred embodiment, the present invention relates to a kind of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine equals, preferably 2-7 doubly to, more preferably about 3 times to, most preferably 3 times combine with described (R)-(+)-the described of Nicotine to described monoclonal antibody, wherein preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.At one more in the highly preferred embodiment, described monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is lower than 80nM, wherein further preferably uses the described monoclonal antibody measuring Kd of IgG2 form; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is lower than 15nM, wherein further preferably use the described monoclonal antibody measuring Kd of IgG2 form, and wherein said monoclonal antibody comprises at least one HCVR, wherein said HCVR comprises: (a) HCCDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1, and wherein preferably described HC CDR1 is positioned at the CDR1 position of the framework of described HCVR; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2, and wherein preferably described HC CDR2 is positioned at the CDR2 position of the framework of described HCVR; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:4, and wherein preferably described HC CDR3 is positioned at the CDR3 position of the framework of described HCVR; And, wherein said monoclonal antibody comprises at least one LCVR, wherein said LCVR comprises: (a) LC CDR1, and wherein said LC CDR1 is made up of the peptide of SEQ ID NO:6, and wherein preferably described LC CDR1 is positioned at the CDR1 position of the framework of described LCVR; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8, and wherein preferably described LC CDR2 is positioned at the CDR2 position of the framework of described LCVR; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of SEQ IDNO:10, and wherein preferably described LC CDR3 is positioned at the CDR3 position of the framework of described LCVR.
Be to be understood that, those skilled in the art will know that how to use specificity from suitable source, to select corresponding LCVR in conjunction with the HCVR of the first antibody of Nicotine, and generation second antibody, wherein said second antibody comprises the described HCVR of described first antibody and the LCVR of selection, and wherein said second antibody can be with the specificity approximately identical with described first antibody in conjunction with Nicotine (" chain reorganization (chain suffling) " be referring to embodiment 9).And it will be understood by those skilled in the art that in a similar fashion, can use the LCVR of first antibody from suitable source, to select corresponding HCVR.Be used to the to increase suitable source of LCVR and/or HCVR is, for example, and from the cDNA of the people experimenter's of the cDNA of natural human B cell, the Nicotine of using by oneself-carrier conjugates immunity B cell, and synthetic library fully, as Morphosys ' HuCAL library.These methods are people such as Kang AS. (Proc Natl AcadSci USA 88,11120-11123,1991), people such as Marks JD. (Biotechnology (N Y) 10,779-783,1992) and people such as Jespers. (Biotechnology (N Y) 12,899-903,1994) have a detailed description in.
Therefore, a HCVR that further aspect is a monoclonal antibody of the present invention, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, and wherein said HCVR comprises: (a) HC CDR1, and wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1; (b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2; (c) HC CDR3, wherein said HC CDR3 is made up of the peptide of any in SEQ ID NO:3 and 4.
In a preferred embodiment, the 7-117 position of described HCVR is made up of the peptide of any among the SEQ ID NO:24,28,33 and 39.
Another aspect of the present invention is the LCVR of monoclonal antibody, wherein said monoclonal antibody is a kind of human monoclonal antibodies, and wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, and wherein said LCVR comprises: (a) LCCDR1, and wherein said LC CDR1 is made up of the peptide of any among the SEQ ID NO:5,6 and 7; (b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8; (c) LC CDR3, wherein said LC CDR3 is made up of the peptide of any in SEQ ID NO:9 and 10.
In a preferred embodiment, the 5-107 position of described LCVR is made up of the peptide of any among the SEQ IDNO:26,31,35,37 and 41.
Further, the present invention relates to a kind of nucleic acid molecule of encode variable region of the present invention, monoclonal antibody of the present invention or its each chain.In a preferred embodiment, described nucleic acid molecule encoding is selected from following peptide: (a) HCVR of the present invention, wherein preferably described HCVR comprise or preferably are made up of the peptide of any among the SEQ ID NO:24,28,33,39,43 and 47; (b) LCVR of the present invention, wherein preferably described LCVR comprise or preferably are made up of the peptide of any among the SEQ ID NO:26,31,35,37,41,45 and 49; (c) single-chain antibody of the present invention, wherein preferably described single-chain antibody comprise or preferably are made up of the peptide of any among the SEQ ID NO:60,62,64,66,68,70 and 72; (d) γ 2 heavy chains of the present invention, wherein preferably described γ 2 heavy chains comprise or preferably are made up of the peptide of any among the SEQ IDNO:73,75,77,80,82 and 84; (e) lambda light chain of the present invention, wherein preferably described lambda light chain comprises or preferably is made up of the peptide of any among the SEQ ID NO:74,76,78,79,81 and 85, (f) κ light chain of the present invention, wherein preferably described κ light chain comprises or preferably is made up of the peptide of SEQ ID NO:83; (g) monoclonal antibody of the present invention.
In a further preferred embodiment, described nucleic acid molecule comprises or is made up of following sequence: the nucleotide sequence of any among the SEQ ID NO:23,27,29,32,38,42 and 46; And/or any nucleotide sequence among the SEQ ID NO:25,30,34,36,40,44 and 48; And/or any nucleotide sequence among the SEQ ID NO:59,61,63,65,67,69 and 71.
Further, the present invention relates to comprise the expression vector of at least a nucleic acid molecule of the present invention.The expression vector that is suitable for expressing monoclonal antibody of the present invention is for example open in PCT/EP2007/061570.
Further, the present invention relates to comprise the host cell of at least a nucleic acid molecule of the present invention or at least a expression vector, wherein preferably described host cell is bacterial cell or eukaryotic cell.In a preferred embodiment, described host cell is to be selected from following eukaryotic cell: (a) yeast cell, (b) insect cell; (c) mammalian cell, wherein preferably described mammalian cell is selected from HEK-293T cell, Chinese hamster ovary celI and COS cell.Most preferably, described mammalian cell is the HEK-293T cell.
Monoclonal antibody of the present invention can be mixed and is suitable in the composition that the experimenter uses.Therefore, further, the present invention relates to a kind of pharmaceutical composition that comprises at least a monoclonal antibody of the present invention, wherein preferably described pharmaceutical composition further comprises pharmaceutically acceptable carrier, thinner or vehicle.Pharmaceutically acceptable carrier, thinner or vehicle be for example at Remington, The Science and Practice of Pharmacy, 19 ThEdition, Gennaro (ed.), Mack publishing Co., Easton, PA, open in 1995.Pharmaceutical composition of the present invention is used with single dose or multiple doses.
In a preferred embodiment, described pharmaceutical composition further comprises at least a additional antibody, and wherein preferably described at least a additional antibody specificity is in conjunction with Nicotine.
Monoclonal antibody of the present invention can be used for passive immunization, is preferably used for people's passive immunization, further preferably is used to resist Nicotine.Monoclonal antibody of the present invention is therefore useful in the treatment of nicotine addiction.Further, the present invention relates to a kind of passive immunization method, preferably resist the passive immunization method of Nicotine, described method comprises pharmaceutical composition of the present invention from significant quantity to the experimenter that use the monoclonal antibody of the present invention or the significant quantity of.
Monoclonal antibody of the present invention and/or pharmaceutical composition preferably are applied to the experimenter, preferably be applied to the people, use standard medicine-feeding technology preferably is selected from oral, intravenous administration, intraperitoneal administration, subcutaneous administration, through lung administration, transdermal administration, intramuscular administration, intranasal administration, orally administering, sublingual administration and suppository administration.
Further, the present invention relates to a kind of method for the treatment of nicotine addiction, described method comprises pharmaceutical composition of the present invention from significant quantity to the experimenter that use the monoclonal antibody of the present invention or the significant quantity of, wherein preferably described experimenter is the people, and wherein further preferably described experimenter is a nicotine addiction.
Further, the present invention relates to be used for the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention of passive immunization, be preferably used for resisting Nicotine, be preferably used for the people, wherein further preferably described monoclonal antibody is applied to described people.
Further, the present invention relates to be used for the treatment of the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention of (preferably people) nicotine addiction.
Further, the present invention relates to monoclonal antibody of the present invention is used for the medicine of passive immunization (preferably resisting Nicotine) in preparation purposes.
Further, the present invention relates to be used for the monoclonal antibody of the present invention of passive immunization (preferably resisting Nicotine).
Further, the present invention relates to purposes in monoclonal antibody of the present invention is used for the treatment of (preferably people) nicotine addiction in preparation the medicine.
Further, the present invention relates to the monoclonal antibody of the present invention in the method for treatment (preferably people) nicotine addiction, used.
Another aspect of the present invention is the application of antibody of the present invention in the method for quantitative and/or qualitative detection Nicotine, preferably detects blood sample, most preferably detects by ELISA.
Should be appreciated that all aspects of the present invention all relate to any monoclonal antibody disclosed herein.
Embodiment
Embodiment 1
Identify the nicotine specific single-chain antibody by mammalian cell displaying method
Use BD Vacutainer TMCPT TMTest tube method (BD Bioscience), separating periphery blood monocytic cell (PBMC) from the volunteer's of inoculation Q β-Nicotine 32ml heparinized blood.Q β of PBMC and Alexa 647nm-mark (3 μ g/ml) and mouse gamma Globulin (10ug/ml; Jackson ImmunoResearch) preincubation, then with following material dyeing: (1) Q β-Nicotine (1 μ g/ml) makes up with the Q β-specificity mouse mAb (1 μ g/ml) of Alexa 488nm-mark and the nicotine specific mouse mAb (1 μ g/ml) of Alexa488nm-mark; (2) the mouse anti human IgM of PE-mark (dilution in 1: 50; BD Biosciences Pharmingen), mouse anti human IgD (dilution in 1: 100; BD Biosciences Pharmingen), mouse anti human CD14 (dilution in 1: 50; BD Biosciences Pharmingen) and mouse anti human CD3 (1: 50 the dilution; BD BiosciencesPharmingen) antibody; (4) the mouse anti human CD19 antibody of PE-texas Red-mark (dilution in 1: 50; Caltag Laboratories).After the dyeing, the washing and filtration cell, and in FACSVantage SE flow cytometer (Becton Dickinson) 443 nicotine specific B of sorting cell (the FL1-positive, FL2-feminine gender, the FL3-positive, FL4-feminine gender).
Substantially as mentioned previously use antigen-specific b cells make up scFv cell surface display library based on sindbis alphavirus (based on the screening of sindbis alphavirus total ask for an interview WO1999/025876A1, PCT/EP2007/061570 is asked for an interview in its application in antibody screening).The cell of showing nicotine specific scFv antibody uses RNase-Nicotine and RNase specificity rabbit polyclonal antibody (2.5 μ g/ml; Abcam) and the anti-rabbit igg antibody of the donkey of FITC-mark (1.5 μ g/ml; Jackson ImmunoResearch) combination separates.With each cell sorting in a hole of 24 orifice plates that contain the 50% BHK feeder cell that converge.The Nicotine combination of the cell that infects is detected by facs analysis, with the virus clone of identification code nicotine specific scFv antibody in virus diffusion back (after the sorting 2 days).
Embodiment 2
The gene rescue of nicotine specific antibody, ELISA screening and order-checking
(referring to application PCT/EP2007/061570) carries out RT-PCR to the supernatant liquor (all containing mono-clonal reorganization sindbis alphavirus) of the bhk cell of the nicotine specific antibody of coding deduction as mentioned previously.The PCR product of the self-contained scFv of each that obtains coding region digests with restriction endonuclease Sfi1, and be cloned among the expression vector pCEP-SP-Sfi-Fc (open as SEQ ID NO:37 in application PCT/EP2007/061570), to allow under the control of CMV promotor, the expressing scFv albumen that merges with the terminal people Fc-of C-γ 1 structural domain.
For elisa assay, use Lipofectamin 2000 (Invitrogen), recommend according to manufacturer, with the form of 24 orifice plates, with each clone's transfection HEK-293T cell.After transfection 2-3 days, collect the supernatant liquor of the scFv-Fc fusion rotein that contains transient expression.In order to check the nicotine specific combination, the Nicotine link coupled RNAse bag that is used in concentration in the phosphate buffered saline buffer (PBS) and is 4 μ g/ml is by elisa plate, and 4 ℃ are spent the night.Abreast, by sandwich method ELISA monitoring scFv-Fc expression level.For this reason, be that the anti-people F of the specific goat of Fc γ (ab ') 2 antibody (the Jackson ImmunoResearch Laboratories 109-006-098) bag of 2.5 μ g/ml is by one group of identical plate with concentration.Use lavation buffer solution (PBS/0.05%Tween) to wash plate then, and at room temperature sealed 2 hours with the 3%BSA in the lavation buffer solution.And then wash plate, and with 3 times of serial dilution incubations of the cell culture supernatant that starts from 1/10 diluent.All dilutions are all carried out in lavation buffer solution.Plate is incubation 2 hours at room temperature, uses the lavation buffer solution thorough washing then.Following then detection bonded scFv-Fc fusion rotein: with Fc γ-anti-human IgG antibody of specificity goat (Jackson ImmunoResearch Laboratories 109-035-098) incubation of HRPO-mark 1 hour.Behind the lavation buffer solution thorough washing, use OPD solution with plate colour developing (1 OPD sheet, 25ml OPD damping fluid and 8 μ l 30%H 2O 2) 5-10 minute, use 5%H 2SO 4The solution termination reaction.Read plate device (Biorad Benchmark) with ELISA then and read plate at OD 450nm.
The ELISA positive colony of coding nicotine specific scFv antibody checks order (referring to application PCT/EP2007/061570) as mentioned previously, comprises clone F018, F063, J004, J042, N049, I022 and N038 (referring to table 2).
The nucleotide sequence of table 2. nicotine specific scFv antibody and the SEQ ID NO of aminoacid sequence
??scFv Nucleotide sequence Aminoacid sequence
??F018 ??59 ??60
??F063 ??61 ??62
??J004 ??63 ??64
??J042 ??65 ??66
??N049 ??67 ??68
??I022 ??69 ??70
??N038 ??71 ??72
Wherein, all variable region of heavy chain comprise VH4 family's family sequence (SEQ ID NO:24,28,33,39,43 and 47).Except that the I022 that comprises V κ 3 sequences (SEQ ID NO:45), variable region of light chain all comprises V λ 1 tame family sequence (SEQ ID NO:26,31,35,37,41 and 49).Importantly, the heavy chain of antibody F018, F063, J004, J042 and N049 is relevant very nearly each other with variable region of light chain, only in several amino acid site difference.The variable region of heavy chain identical (SEQ ID NO:28) of clone F063 and J042.The nucleotide sequence and being summarised in the table 3 of aminoacid sequence that comprise about the variable region of Nicotine binding antibody provide.
The SEQ ID NO of nucleotide sequence that table 3. nicotine specific antibody HCVR and LCVR comprise and aminoacid sequence
??scFv Nucleotide sequence (HCVR) 1 Aminoacid sequence (HCVR) Nucleotide sequence (LCVR) 2 Aminoacid sequence (LCVR)
??F018 ??23 ??24 ??25 ??26
??F063 ??27 ??28 ??30 ??31
??J004 ??32 ??33 ??34 ??35
??J042 ??29 ??28 ??36 ??37
??N049 ??38 ??39 ??40 ??41
??I022 ??42 ??43 ??44 3 ??45
??N038 ??46 ??47 ??48 ??49
1)All HCVR comprise the VH4 sequence;
2)Except the LCVR of I022, all LCVR comprise V λ 1 sequence;
3)The LCVR of I022 comprises V κ 3 sequences.
Embodiment 3
Nicotine specific scFv-Fc Expression of Fusion Protein and purifying
Being expressed on a large scale in the HEK-293T cell of scFv-Fc fusion rotein carried out.Transfection the day before yesterday is for every kind of protein will expressing, with 5x10 6Individual 293T cell inoculation is to 10cm tissue culturing plate.Use Lipofectamin Plus (Invitrogen) then, recommend,, and be inoculated into again on the 14cm culture dish of existence 1 a μ g/ml tetracycline with corresponding scFv-Fc fusion constructs transfectional cell, incubation 1 day according to manufacturer.Select after 3 days the tetracycline resistant cell to be transferred on three 14cm plates with poly-L-Lysine bag quilt, and grow to and converge.Then substratum is replaced with serum free medium, collected the supernatant liquor that contains corresponding scFv-Fc fusion rotein in per 3 days, and filter by 0.22 μ M Millex GV sterile filters (Millipore).
For every kind of scFv-Fc fusion rotein, continuous cutting is combined, and be added on albumin A-sepharose post.Phosphate buffered saline buffer (PBS) with 10 times of column volumes is washed post, bonded protein 0.1M glycine pH 3.6 wash-outs.The 1ml fraction is collected in contains in the test tube that is useful on neutral 0.1ml 1M Tris pH 7.5.Contain the protein fraction by SDS-PAGE analysis and merging.By using 10'000MWCO Slide-A-Lyzer dialysis cassette (Pierce) dialysis that buffer exchange is PBS.Then the protein purification among the PBS is filtered by 0.22 μ MMillex GV sterile filters (Millipore), and five equilibrium.The stock solution of will working is kept under 4 ℃, and the equal portions of long storage are freezing fast in liquid nitrogen, and is kept under-80 ℃.
Embodiment 4
By suppressing the combination of ELISA checking nicotine specific
In order to get rid of the possible joint specificity of antibody, confirm and the combining of the Nicotine that dissociates by suppressing ELISA.Therefore, Nicotine-link coupled RNAse that it is 4 μ g/ml that elisa plate is used in the middle concentration of phosphate buffered saline buffer (PBS) wraps quilt, and 4 ℃ are spent the night.Use lavation buffer solution (PBS/0.05%Tween) to wash plate then, and at room temperature with the sealing of the 3%BSA in the lavation buffer solution 2 hours.And then wash plate, under the situation of the free Nicotine (0.1-100 μ M) that exists or do not exist concentration to improve gradually, with concentration be scFv-F018, scFv-F063, scFv-I022, scFv-J004, scFv-N038 and the scFv-N049 incubation of the purifying of 100ng/ml.Plate is incubation 2 hours at room temperature, uses the lavation buffer solution thorough washing then.Following then detection bonded scFv-Fc fusion rotein: with Fc γ-anti-human IgG antibody of specificity goat (Jackson ImmunoResearch Laboratories 109-035-098) incubation of HRPO-mark 1 hour.Behind the lavation buffer solution thorough washing, make plate colour developing (1 OPD sheet, 25ml OPD damping fluid and 8 μ l 30%H with OPD solution 2O 2) 5-10 minute, and use 5%H 2SO 4The solution termination reaction.Read plate device (Biorad Benchmark) with ELISA then and read plate at OD 450nm.Every kind of antibody effectively suppresses with combining by the free Nicotine of fixed Nicotine, and the IC50 value is in low micro-molar range.
Embodiment 5
Structure, expression and the purifying of people's nicotine specific IgG2
Use variable region transspecific primer (open as SEQ ID NO:92-103 in application PCT/EP2007/061570), by heavy chain and the variable region of light chain coding section of pcr amplification scFv-F018, scFv-F063, scFv-I022, scFv-J004, scFv-J042, scFv-N038 and scFv-N049.For example, use the DNA of the variable region of light chain of primer VL-Sac1-F4 (5 '-CAG GCG GCC GAGATC GAG CTC ACT CAG C-3 ') and VL-EcoR5-B1 (5 '-ACC GCC GAGGAT ATC CAG CTG GGT-3 ') amplification coding antibody F018.Use the DNA of primer VH-XhoI-F (5 '-SAG GTG CAG CTG CTC GAG TCKGG-3 ') and VH-ApaI-B (5 '-GCC ACT AGT GAC CGA TGG GCC-3 ') amplification coding variable region of heavy chain.The DNA of other light chain and variable region of heavy chain of encoding correspondingly increases.
λ that obtains or kappa light chain variable district PCR product digest with restriction enzyme SacI and EcoR5, by the agarose gel electrophoresis purifying, and be connected respectively among the pCMV-LC-λ or pCMV-LC-κ of SacI-EcoR5 digestion (in application PCT/EP2007/061570 as SEQID NO:110 and 71 open).The carrier that obtains allows people's light chain of The expressed, comprises signal peptide, for example F018 lambda light chain (SEQ ID NO:56).Similarly, variable region of heavy chain PCR product digests with restriction enzyme XhoI and ApaI, gel-purified, and be connected to the pCMV-HC-γ 2 interior (open as SEQ ID NO:78 in application PCT/EP2007/061570) that XhoI-ApaI digests.The carrier that obtains allows people γ 2 heavy chains of The expressed, comprises signal peptide, for example F018 γ 2 heavy chains (SEQ ID NO:55).
PCMV-LC-λ or-the κ expression construct can produce complete IgG2 in principle with the coexpression of corresponding pCMV-HC-γ 2 expression construct separately.But,, at first heavy chain and light chain coding region are combined to (open as SEQID NO:104 in application PCT/EP2007/061570) among the expression vector pCB15 based on EBNA in order to improve productive rate and to help large-scale antibody producing.For example, for antibody F018 is expressed as complete IgG2, by downcutting γ 2 heavy chain coding regions from pCMV-F018-HC-γ 2 with restriction enzyme AscI and PacI digestion, the 1403bp fragment that obtains is by the agarose gel electrophoresis purifying, be connected to then among the pCB15 of Asc1-Pac1 digestion, produce pCB15-F018-HC-γ 2.Then by downcutting F018 lambda light chain coding region from pCMV-F018-LC-λ with Nhe1 and Pme1 digestion, the 723bp fragment that gel-purified obtains, be connected to then in the pCB15-F018-HC-γ 2 of Nhe1-Pme1 digestion, produce complete IgG expression vector pCB15-F018-IgG2 λ.As (embodiment 2) as described in for the scFv-Fc fusion rotein, in the HEK-293T cell, carry out the expression of complete IgG fully.
Embodiment 6
Structure, expression and the purifying of complete people's nicotine specific IgG2
Mainly the synthesizing of complete IgG2 molecule of people as the pCB15 expression vector guiding of structure as described in the embodiment 4.But, compare with fully human antibodies, still there are two small differences.The first, the sequence of heavy chain or light chain signal peptide is by expression vector codes, and generally do not correspond to the natural signals peptide of described variable region.The second, the amino acid sites that is positioned at heavy chain or variable region of light chain flank is predetermined by carrier, for example, is owing to there is restriction site.Therefore, in order to produce complete people mAb, these differences of each in needs correction heavy chain and the light chain expression vector.For example, guiding comprises the following generation of carrier that complete people F018 γ 2 heavy chains (SEQ IDNO:57) of natural signals peptide are expressed.At first use primers F 018-HC-fh-F1 (5 '-TTCCTC CTG TTG GTG GCA GCA CCC AGG TGG GTG CTG TCC CAGCTG CAA CTG CAG GAG TC-3 ') and F018-HC-fh-B1 (5 '-GAC CGA TGGGCC CTT GGT GGA AGC-3 '), by PCR from plasmid pCMV-F018-HC-γ 2 amplification F018 variable region of heavy chain.The PCR product that obtains uses primers F 018-HC-fh-F2 (5 '-GAG GGC GCG CCA CCA TGA AGC ACC TGT GGT TCT TCCTCC TGT TGG TGG CAG C-3 ') and F018-HC-fh-B1 further to increase then.The PCR end product, by the agarose gel electrophoresis purifying and is connected in the pCMV-HC-γ 2 of Asc1-Apa1 digestion with restriction enzyme Asc1 and Apa1 digestion, produces plasmid pCMV-fhF018-HC-γ 2.
Similarly, guiding comprises the following generation of carrier that the complete people F018 lambda light chain (SEQ IDNO:58) of natural signals peptide is expressed.At first, use primers F 018-LC-fh-F1 (5 '-CCTCCT CAC CCT CCT CAC TCA CTG CGC CGG GTC CTG GGC CCAGTC TGT GCT CAC-3 ') and F018-LC-fh-B1 (5 '-GGG CTG ACC TAG CACGGT CAG CTG GGT GCC-3 '), by PCR from the pCMV-F018-LC-λ F018 variable region of light chain that increases.Abreast, use primers F 018-LC-fh-F3 (5 '-GGC ACCCAG CTG ACC GTG CTA GGT CAG CCC-3 ') and T7 (5 '-TAA TAC GACTCA CTA TAG GG-3 ') amplification lambda light chain.Secondly, the PCR product that uses primers F 018-LC-fh-F2 (5 '-GAG GCT AGC GCC ACC ATG GCC GGC TTC CCC CTC CTC CTCACC CTC CTC ACT C-3 ') and T7 to obtain by the PCR assembling.The PCR end product, by the agarose gel electrophoresis purifying and is connected in the pCMV-LC-κ of Nhe1-Pme1 digestion with restriction enzyme Nhe1 and Pme1 digestion, produces plasmid pCMV-fhF018-LC-λ.
Use 4 described clone's strategies,, make up the carrier pCB15-fhF018-IgG2 λ that the complete people mAb F018 of guiding expresses by heavy chain and light chain coding region are cloned in the pCB15 successively as embodiment.The expression vector that guides complete people mAbs F063, I022, J004, J042, N038 and N049 to express makes up in a similar fashion.
Embodiment 7
Determine avidity by equilibrium dialysis
Use DispoEquilibriumDialyzer (Harvard Biosciences), measure the avidity of antibody (S)-(-)-Nicotine by equilibrium dialysis.Two chambers of dialysis apparatus separate with film (10'000Da cutoff value).Chamber is filled in scFv-Fc or the IgG2 that concentration among the PBS/2%BSA is respectively the purifying of 5.4 μ g/ml or 7.5 μ g/ml, and another chamber does not contain antibody.Working concentration is tritium-labeled (S)-(-)-Nicotine (Amersham) of 3-444nM.Allow equilibrium dialysis carry out 48-72 hour.From two side-draw equal portions of chamber, and in scintillometer, measure radioactivity.Deducting the radioactivity of the chamber that do not contain antibody measuring corresponding to the radioactivity of (S)-(-)-Nicotine of antibodies by the radioactivity of measuring with the chamber that contains antibody calculates.Determine free (S)-(-)-nicotine concentration by the radioactivity that the chamber that does not contain antibody is measured.By with the binding radioactivity of single-point combination model match, obtain equilibrium dissociation constant (Kd) as the mensuration of the function of free (S)-(-)-nicotine concentration.Most of nicotine specific antibody with the Kd value of low nmole scope in conjunction with free (S)-(-)-Nicotine (table 4).The avidity and the scFv-Fc of IgG2 antibody are suitable, and the Kd value differs and is no more than about 2 times.IgG2-F018 has the dissociation constant of 7nM, and almost the value of measuring than scFv-Fc-F018 is low 2 times, than low 4 times at least of the Kd of another IgG2.Avidity that it should be noted that anti-(S)-(-)-Nicotine polyclonal antibody is 30-70nM, promptly be significantly less than clone F018 avidity (people such as Maurer P.., 2005, Eur.J.Immunol.35,2031-2040).
The dissociation constant Kd value (nM) that table 4. is measured by equilibrium dialysis.Except only measuring IgG2-N038 once, shown value is the mean value ± SD of at least 2 independent experiments.
Form ??F018 ??F063 ??I022 ??J004 ??J042 ??N038 ??N049
??scFv-Fc ??11.5±1.5 ??70.5±14 ??42.5±17 ??33.3±2 ??47.0±3.1 ??686.3±58 ??72.7±5.6
??IgG2 ??7.4±1.2 ??31.4±13 ??89.9±9.8 ??64.5±2.2 ??30.2±11 ??743.8 ??35.6±3.5
The cross reactivity of mAb IgG2-F018 and (-)-cotinine and vagusstoff is total to incubation by the 10 times of serial dilutions (1nM-100 μ M) with these compounds with tritium-labeled (S)-(-) of 56nM-Nicotine and determines (Fig. 1) in aforesaid equilibrium dialysis chamber.As expection, unlabelled (-)-Nicotine is replaced tritium-labeled (S)-(-)-Nicotine easily.On the contrary, structurally irrelevant with Nicotine but combine the identical combination bag of Nicotine acetylcholine receptor vagusstoff (people such as CelieP.H.., 2004, Neuron 41,907-914) even also do not disturb the combination of (S)-(-)-Nicotine when 4000 times of molar excess.Similarly, as the main metabolites of (S)-(-)-Nicotine and (-)-cotinine (Benowitz N.L. of in smoker's blood, existing with respect to Nicotine 10-20 times of molar excess, 1997, Br.J.Clin.Pharmacol.43 259-267) can not combine IgG2-F018 with (S)-(-)-Nicotine effective competition.Based on the IC50 that observes, can estimate IgG2-F018 and the binding ratio of (-)-cotinine and combine weak about 1000 times of (S)-(-)-Nicotine.Therefore, the impossible result of treatment of disturbing IgG2-F018 of the existence of (-)-cotinine of molar excess in smoker's blood.
We next measured mAbs IgG2-F018 ,-F063 ,-J004 ,-J042 and-N049 is to the avidity of (R)-(+)-isomer of Nicotine.This steric isomer is present in the tobacco plant.In addition, the combustion processes when it can be by smoking forms, and to central nervous system have the activity similar in nature to (S)-(-)-Nicotine (people such as Hukkanen J.., 2005, Pharmacol.Rev.57,79-115).Therefore, in the presence of unlabelled (R)-(+)-Nicotine (1nM-100 μ M) that concentration improves gradually, detect tritium-labeled (S)-(-)-Nicotine (F018:56nM of fixed concentration by equilibrium dialysis; F063, J004, J042, N049:111nM) with the combining of every kind of antibody.By using the match of single-point competitive model to obtain equilibrium dissociation constant (Kd) as the binding radioactivity of the mensuration of the function of (R)-(+)-nicotine concentration.Surprisingly, (R)-(+)-Nicotine is very effective aspect (S)-(-)-Nicotine competition combines every kind of antibody, and its Kd value is than approximately low 2-5 times (table 5) of (S)-(-)-Nicotine.
The antibody that table 5. is measured by equilibrium dialysis-(R)-(+)-the interactional dissociation constant Kd of Nicotine (nM)
Form ??F018 ??F063 ??J004 ??J042 ??N049
??IgG2 ??3.1 ??6.6 ??12.6 ??7.7 ??10.5
Embodiment 8
The evaluation that Nicotine distributes in mice plasma and the brain
Respectively organize in the mouse peritoneum the various recombinant human IgG2 monoclonal antibodies of injection 0.1-3mg by what the only female Balb/c mouse of 5-6 was formed.After one day, by analyzing the inhibition that Nicotine enters brain to tritium-labeled (S)-(-) of tail intravenous injection-Nicotine (Amersham) (375,750 or 1500ng).Pass through CO after 5 minutes 2Suffocate and kill mouse, and gather blood and brain.By the nicotine concentration in radioactivity calculating serum that exists and the brain.Blood content according to brain is proofreaied and correct brain nicotine concentration (3 μ l/100mg).With respect to the nicotine concentration of in the mouse brain of injection contrast IgG, finding, calculate the reduction per-cent of the Nicotine picked-up in the brain.For all nicotine specific antibody that detect, observe the minimizing that Nicotine distributes in brain.For most of antibody, observed minimizing is generally 40% or higher when dosage is every mouse 0.5mg.The strongest mAb is F018 under these experiment conditions, and Nicotine entering in brain always reduced (table 6 and 7) more than 60%.
Table 6. nicotine specific antibody (uses 0.5mg IgG2 passive immunization to the inhibition that Nicotine enters mouse brain; Use the 750ng Nicotine to attack)
Antibody Brain Nicotine (cpm/g) % reduces
Contrast IgG ??131559±14650 ??-
??F018 ??46717±3550 ??64
??J004 ??77002±9293 ??41
??J042 ??72678±7423 ??45
??N049 ??68395±2885 ??48
Table 7. nicotine specific antibody (uses 0.5mg IgG2 passive immunization to the inhibition that Nicotine enters mouse brain; Use the 750ng Nicotine to attack)
Antibody Brain Nicotine (cpm/g) % reduces
Contrast IgG ??114048±13786 ??-
??F018 ??42803±3751 ??62
??F063 ??54783±3909 ??52
??I022 ??68687±5498 ??40
??N038 ??91823±14311 ??19
Embodiment 9
Use the variable region of heavy chain of anti-Nicotine antibody F018 to identify complementary variable region of light chain (" chain reorganization ")
Do not provide the nucleotide sequence of all primers sequence, that use in the present embodiment can be herein at Phage Display-A Laboratory Manual, Cold Spring HarborLaboratory Press, 2001, under identical title, find among the Protocol 9.2.
Use primer HSCVH35-FL and HSC-F018-B (5 '-CCT GGC CGG CCTGGC CAC TAG TGA CCG ATG GGC CCT TGG TGG AAG C-3 ', SEQID NO:86), by the dna sequence dna of PCR from the variable region of heavy chain of the dna molecular amplification coding antibody F018 that comprises scFv-F018 sequence (SEQ ID NO:59).Use 4 kinds have adopted primer HSCK1-F, HSCK24-F, HSCK3-F and HSCK5-F etc. molar mixture, add 4 kinds of antisense primer HSCJK14o-B, HSCJK2o-B, HSCJK3o-B and HSCJK5o-B etc. molar mixture, by PCR from library from the dna sequence dna of the cDNA amplification coding human kappa light chain variable region of the people experimenter's of Nicotine-carrier conjugates immunity B cell.Use 9 kinds have adopted primer HSCLam1a, HSCLam1b, HSCLam2, HSCLam3, HSCLam4, HSCLam6, HSCLam78, HSCLam9 and HSCLam10 etc. molar mixture, add 3 kinds of antisense primer HSCJLam1236, HSCJLam4 and HSCJLam57 etc. molar mixture, by PCR from library from the dna sequence dna in the cDNA amplification coding people lambda light chain variable district of the people experimenter's of Nicotine-carrier conjugates immunity B cell.
In order to select complementary variable region of light chain, use primer RSC-F and RSC-B, by the overlapping extension of PCR of F018 VH PCR product and V κ library PCR product or V λ library PCR product, assembling scFv coding region.5 ' variable region of light chain library (κ or λ) and the 3 ' variable region of heavy chain of the about 750-800bp PCR product coding nicotine specific antibody F018 that obtains, this variable region of light chain is connected by 18 amino acid whose flexible joints with variable region of heavy chain, and flank is two SfiI restriction sites.ScFv-κ and-λ library fragment mixes to wait molar ratio, digest with restriction endonuclease SfiI, and be cloned in the carrier pDel-SP-TM, be used for as described in embodiment 1, screening substantially by scFv cell surface display method based on sindbis alphavirus.Perhaps, the pComb3H that the scFv library fragment cloning of SfiI-digestion is digested to SfiI-(Rader C, Barbas CF 3rd., 1997, Curr Opin Biotechnol.8,503-508) in.Expressing antibodies then, and use described program to select by the phage display method, be used in conjunction with fixed RNAse-Nicotine conjugate (Phage Display:A Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001).
Sequence table
SEQ?ID?NO：1?HCDR1-F018/F063/J004/J042/N049
GGSI
 
SEQ?ID?NO：2?HCDR2-F018/F063/J004/J042/N049
IYSSGST
 
SEQ?ID?NO：3?HCDR3-F018
VAWFGDLLSLKGVEL
 
SEQ?ID?NO：4?HCDR3-F063/J004/J042/N049
VVWFGDLLSLKGVEL
 
SEQ?ID?NO：5?LCDR1-F018/J042
SSNIGSNY
 
SEQ?ID?NO：6?LCDR1-F063/N049
SSNIGSKN
 
SEQ?ID?NO：7?LCDR1-J004
SSNIGSSY
 
SEQ?ID?NO：8?LCDR2-F018/F063/J004/J042/N049
RNN
 
SEQ?ID?NO：9?LCDR3-F018
AAWDDSLSAWV
 
SEQ?ID?NO：10?LCDR3-F063/J004/J042/N049
AAWDDSLSGWV
 
SEQ?ID?NO：11?HCDR1-I022
GGSISSTSYY
 
SEQ?ID?NO：12?HCDR2-I022
ISYSGST
 
SEQ?ID?NO：13?HCDR3-I022
ARILWFGEYLGDY
 
SEQ?ID?NO：14?LCDR1-I022
QSVSNY
 
SEQ?ID?NO：15?LCDR2-I022
GAS
 
SEQ?ID?NO：16?LCDR3-I022
QQYYSTPWT
 
SEQ?ID?NO：17?HCDR1-N038
GYSISSGYY
 
SEQ?ID?NO：18?HCDR2-N038
SNHSGST
 
SEQ?ID?NO：19?HCDR3-N038
AREAGYSSSWYFDY
 
SEQ?ID?NO：20?LCDR1-N038
SSNIGNNY
 
SEQ?ID?NO：21?LCDR2-N038
DNN
 
SEQ?ID?NO：22?LCDR3-N038
GTWDSSLSAWV
 
SEQ ID NO:23 VH-F018 (117 aa7-117, NA)
TCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCT
CCATCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTC
TAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCACATCCGTAGACACGTCC
AAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTG
TGGCGTGGTTCGGGGACTTATTATCGTTGAAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCA
 
SEQ ID NO:24 VH-F018 (117 aa7-117, AA)
SGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVTTSVDTS
KNQFSLRLSSVTAADTAVYYCVAWFGDLLSLKGVELWGQGTLVTVSS
 
SEQ ID NO:25 VL-F018 (110 aa5-107, NA)
ACTCAGCCACCTTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCA
GCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACT
CCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTG
CAGCATGGGATGACAGCCTGAGTGCTTGGGTGTTCGGCGGAGGCACCCAGCTG
 
SEQ ID NO:26 VL-F018 (110 aa5-107, AA)
TQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKS
GTSASLAISGLRSEDEADYYCAAWDDSLSAWVFGGGTQL
 
SEQ ID NO:27 VH-F063 (117 aa7-117, NA)
TCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCT
CCATCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTC
TAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCC
AAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTG
TGGTGTGGTTCGGGGACTTATTATCGTTGAAGGGGGTCGAATTGTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCA
 
SEQ ID NO:28 VH-F063-J042 (117 aa7-117, AA)
SGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVTISVDTS
KNQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSS
SEQ ID NO:29 VH-J042 (117 aa7-117, NA)
TCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCT
CCATCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTC
TAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCC
AAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTG
TGGTGTGGTTCGGGGACTTATTATCGTTGAAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCA
 
SEQ ID NO:30 VL-F063 (110 aa5-107, NA)
ACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCACCGTCTCTTGTTCTGGAAGCA
GCTCTAACATCGGAAGTAAAAATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACT
CCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTG
CAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCAGCGGAGGCACCAAGGTG
 
SEQ ID NO:31 VL-F063 (110 aa5-107, AA)
TQPPSVSGTPGQRVTVSCSGSSSNIGSKNVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKS
GTSASLAISGLRSEDEADYYCAAWDDSLSGWVFSGGTKV
 
SEQ ID NO:32 VH-J004 (117 aa7-117, NA)
TCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCACACCTGCACTGTCTCTGGTGGCT
CCATCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTC
TAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCC
AAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTG
TGGTGTGGTTCGGGGACTTATTATCGTTGAAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCA
 
SEQ ID NO:33 VH-J004 (117 aa7-117, AA)
SGPGLVKPSETLSHTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVTISVDTS
KNQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSS
 
SEQ ID NO:34 VL-J004 (110 aa5-107, NA)
ACGCAGCCGCCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCA
GCTCCAACATCGGAAGTAGTTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACT
CCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTG
CAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGCACCGAGCTG
 
SEQ ID NO:35 VL-J004 (110 aa5-107, AA)
TQPPSVSGTPGQRVTISCSGSSSNIGSSYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKS
GTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTEL
 
SEQ ID NO:36 VL-J042 (110 aa5-107, NA)
ACCCAGGAGCCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCA
GCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACT
CCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGCCCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTG
CAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTG
SEQ ID NO:37 VL-J042 (110 aa5-107, AA)
TQEPSVSGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGAPDRFSGSKS
GTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKL
 
SEQ ID NO:38 VH-N049 (117 aa7-117, NA)
TCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGTT
CCATCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTC
TAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCC
GAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTG
TGGTGTGGTTCGGGGACTTATTATCGTTGAAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCA
 
SEQ ID NO:39 VH-N049 (117 aa7-117, AA)
SGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVTISVDTS
ENQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSS
 
SEQ ID NO:40 VL-N049 (110 aa5-107, NA)
ACTCAGCCACCCACAGTGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCA
GCTCCAACATCGGAAGTAAAAATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACT
CCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTG
CAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGCACCAAGGTG
 
SEQ ID NO:41 VL-N049 (110 aa5-107, AA)
TQPPTVSGTPGQRVTISCSGSSSNIGSKNVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKS
GTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKV
 
SEQ ID NO:42 VH-I022 (121 aa7-121, NA)
TCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCT
CCATCAGCAGTACTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTG
GATTGGGAGTATCTCTTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACC
ATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCAGACA
CGGCTGTGTATTACTGTGCGAGGATACTATGGTTCGGAGAGTACCTAGGGGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCA
 
SEQ ID NO:43 VH-I022 (121 aa7-121, AA)
SGPGLVKPSETLSLTCTVSGGSISSTSYYWGWIRQPPGKGLEWIGSISYSGSTYYNPSLKSRVT
ISVDTSKNQFSLKLTSVTAADTAVYYCARILWFGEYLGDYWGQGTLVTVSS
 
SEQ ID NO:44 VL-I022 (107 aa5-104, NA)
ACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCA
GTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCT
CATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGG
ACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATCACTGTCAGC
AATATTATAGTACTCCGTGGACGTTCGGCCAAGGGACCAAAGTG
 
SEQ ID NO:45 VL-I022 (107 aa5-104, AA)
TQSPGTLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYHCQQYYSTPWTFGQGTKV
SEQ ID NO:46 VH-N038 (121 aa7-121, NA)
TCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACT
CCATCAGCAGTGGTTACTACTGGGGCTGGATCCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGAT
TGGGAGTAGCAATCATAGTGGGAGCACCTACTACAACCCGTCCCTCAGGAGTCGAGTCACCATA
TCAGTAGACACGTCCAAGAACCAATTCTCCCTGAAGGTGAACTCTGTGACCGCCGCAGACACGG
CCGTTTATTACTGTGCGAGAGAGGCGGGGTATAGCAGCAGCTGGTACTTTGACTACTGGGGTCA
GGGAACCCTGGTCACCGTCTCCTCA
 
SEQ ID NO:47 VH-N038 (121 aa7-121, AA)
SGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIGSSNHSGSTYYNPSLRSRVTI
SVDTSKNQFSLKVNSVTAADTAVYYCAREAGYSSSWYFDYWGQGTLVTVSS
 
SEQ ID NO:48 VL-N038 (110 aa5-107, NA)
ACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCA
GCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACT
CCTCATTTATGACAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCG
GAACATGGGATAGCAGCCTGAGTGCTTGGGTGTTCGGCGGAGGGACCCAGCTG
 
SEQ ID NO:49 VL-N038 (110 aa5-107, AA)
TQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKS
GTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTQL
 
SEQ ID NO:50 VH (aa1-6, all preferred sequence, AA)
QX1QLQX2, wherein X1 is selected from V and L, and wherein X2 is selected from E and Q
 
SEQ ID NO:51 VL (aa1-4, F018, F063, J004, J042, N049, the preferred sequence of N038, AA)
QSVL
 
SEQ ID NO:52 VL (aa1-4, the preferred sequence of I022, AA)
EIVX1, wherein X1 is selected from L and M
 
SEQ ID NO:53 VL (aa108-110, F018, F063, J004, J042, N049, the preferred sequence of N038, AA)
TVL
 
SEQ ID NO:54 VL (aa105-107, the preferred sequence of I022, AA)
X1IK, wherein A1 is selected from D and E
 
SEQ ID NO:55 F018 γ 2 heavy chains (having flanking sequence and signal peptide)
MDWTWRILFLVAAATGAHSQMQLLESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIG
SIYSSGSTYYNPSLKSRVTTSVDTSKNQFSLRLSSVTAADTAVYYCVAWFGDLLSLKGVELWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV
LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK
SEQ ID NO:56 F018 lambda light chain (having flanking sequence and signal peptide)
MAWALLLLTLLTQGTGSWAQSELTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAP
KLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSAWVFGGGTQLDI
LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN
NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
The complete people F018 of SEQ ID NO:57 γ 2 heavy chains (having flanking sequence and signal peptide)
MKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIG
SIYSSGSTYYNPSLKSRVTTSVDTSKNQFSLRLSSVTAADTAVYYCVAWFGDLLSLKGVELWGQ
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV
LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK
 
The complete people F018 of SEQ ID NO:58 lambda light chain (having flanking sequence and signal peptide)
MAGFPLLLTLLTHCAGSWAQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAP
KLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSAWVFGGGTQLTV
LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN
NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
SEQ ID NO:59 scFv-F018 Sfi1-fragment (NA)
NGGCCCAGGCGGCCGAGCTCGTGCTGACTCAGCCACCTTCAGCGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAG
CAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCC
CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTC
CGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGCTTGGGTGTTCGGC
GGAGGCACCCAGCTGACCGTCCTCGGTGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
GCTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGCAGTCTGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCTGGGGCTGGATCCGCCAGCCCCCA
GGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTCTAGTGGGAGCACCTACTACAACCCGTCCC
TCAAGAGTCGAGTCACCACATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGAGCTC
TGTGACCGCCGCAGACACGGCTGTGTATTACTGTGTGGCGTGGTTCGGGGACTTATTATCGTTG
AAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCC
CATCGGTCACTAGTGGCCAGGCCGGCCN
 
SEQ ID NO:60 scFv-F018 Sfi1-fragment (AA)
XAELVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDR
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSAWVFGGGTQLTVLGGGSSRSSSSGGGGSG
GGGEVQLVQSGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKS
RVTTSVDTSKNQFSLRLSSVTAADTAVYYCVAWFGDLLSLKGVELWGQGTLVTVSSASTKGPSV
TSGQA
 
SEQ ID NO:61 scFv-F063 Sfi1-fragment (NA)
NGGCCCAGGCGGCCGAGCTCGTGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAG
GGTCACCGTCTCTTGTTCTGGAAGCAGCTCTAACATCGGAAGTAAAAATGTATACTGGTACCAG
CAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCC
CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTC
CGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCAGC
GGAGGCACCAAGGTGACCGTCCTAGGTGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
GCTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCTGGGGCTGGATCCGCCAGCCCCCA
GGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTCTAGTGGGAGCACCTACTACAACCCGTCCC
TCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGAGCTC
TGTGACCGCCGCAGACACGGCTGTGTATTACTGTGTGGTGTGGTTCGGGGACTTATTATCGTTG
AAGGGGGTCGAATTGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCC
CATCGGTCACTAGTGGCCAGGCCGGCCN
 
SEQ ID NO:62 scFv-F063 Sfi1-fragment (AA)
XAELVLTQPPSVSGTPGQRVTVSCSGSSSNIGSKNVYWYQQLPGTAPKLLIYRNNQRPSGVPDR
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFSGGTKVTVLGGGSSRSSSSGGGGSG
GGGEVQLVESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKS
RVTISVDTSKNQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSSASTKGPSV
TSGQA
 
SEQ ID NO:63 scFv-J004 Sfi1-fragment (NA)
NGGCCCAGGCGGCCGGGCTCGTGGTGACGCAGCCGCCCTCAGTGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAGTTATGTATACTGGTACCAG
CAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCC
CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTC
CGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGC
GGAGGCACCGAGCTGACCGTCCTCGGTGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
GCTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCACACCTGCACTGTCTCTGGTGGCTCCATCTGGGGCTGGATCCGCCAGCCCCCA
GGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTCTAGTGGGAGCACCTACTACAACCCGTCCC
TCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGAGCTC
TGTGACCGCCGCAGACACGGCTGTGTATTACTGTGTGGTGTGGTTCGGGGACTTATTATCGTTG
AAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCC
CATCGGTCACTAGTGGCCAGGCCGGCCN
 
SEQ ID NO:64 scFv-J004 Sfi1-fragment (AA)
XAGLVVTQPPSVSGTPGQRVTISCSGSSSNIGSSYVYWYQQLPGTAPKLLIYRNNQRPSGVPDR
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTELTVLGGGSSRSSSSGGGGSG
GGGEVQLVESGPGLVKPSETLSHTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKS
RVTISVDTSKNQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSSASTKGPSV
TSGQA
 
SEQ ID NO:65 scFv-J042 Sfi1-fragment (NA)
NGGCCCAGGCGGCCGAGCACGTGGTGACCCAGGAGCCCTCAGTGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAG
CAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGCCC
CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTC
CGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGC
GGAGGGACCAAGCTGACCGTCCTAGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
GCTCGGGCGGTGGTGGGCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCTGGGGCTGGATCCGCCAGCCCCCA
GGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTCTAGTGGGAGCACCTACTACAACCCGTCCC
TCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGAGCTC
TGTGACCGCCGCAGACACGGCTGTGTATTACTGTGTGGTGTGGTTCGGGGACTTATTATCGTTG
AAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCC
CATCGGTCACTAGTGGCCAGGCCGGCCN
 
SEQ ID NO:66 scFv-J042 Sfi1-fragment (AA)
XAEHVVTQEPSVSGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGAPDR
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGGGSSRSSSSGGGGSG
GGGQVQLQESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKS
RVTISVDTSKNQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSSASTKGPSV
TSGQA
 
SEQ ID NO:67 scFv-N049 Sfi1-fragment (NA)
NGGCCCAGGCGGCCGAGCTCGTGCTGACTCAGCCACCCACAGTGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAAAAATGTATACTGGTACCAG
CAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCC
CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTC
CGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGC
GGAGGCACCAAGGTGACCGTCCTAGGTGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
GCTCGGGCGGTGGTGGGCAGATCACCTTGAAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGTTCCATCTGGGGCTGGATCCGCCAGCCCCCA
GGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTCTAGTGGGAGCACCTACTACAACCCGTCCC
TCAAGAGTCGAGTCACCATATCCGTAGACACGTCCGAGAACCAGTTCTCCCTGAGGCTGAGCTC
TGTGACCGCCGCAGACACGGCTGTGTATTACTGTGTGGTGTGGTTCGGGGACTTATTATCGTTG
AAGGGGGTTGAATTGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCC
CATCGGTCACTAGTGGCCAGGCCGGCCN
 
SEQ ID NO:68 scFv-N049 Sfi1-fragment (AA)
XAELVLTQPPTVSGTPGQRVTISCSGSSSNIGSKNVYWYQQLPGTAPKLLIYRNNQRPSGVPDR
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKVTVLGGGSSRSSSSGGGGSG
GGGQITLKESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKS
RVTISVDTSENQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSSASTKGPSV
TSGQA
 
SEQ ID NO:69 scFv-I022 Sfi1-fragment (NA)
NGGCCCAGGCGGCCGAGCTCCACATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAGCAG
AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAG
ACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGA
AGATTTTGCAGTGTATCACTGTCAGCAATATTATAGTACTCCGTGGACGTTCGGCCAAGGGACC
AAAGTGGATATCAAAGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTG
GTGGGCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCT
CACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTACTAGTTACTACTGGGGCTGGATCCGCCAG
CCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTCTTATAGTGGGAGCACCTACTACAACC
CGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCT
GACCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGGATACTATGGTTCGGAGAG
TACCTAGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC
CATCGGTCACTAGTGGCCAGGCCGGCCN
 
SEQ ID NO:70 scFv-I022 Sfi1-fragment (AA)
XAELHMTQSPGTLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRF
SGSGSGTDFTLTISRLEPEDFAVYHCQQYYSTPWTFGQGTKVDIKGGSSRSSSSGGGGSGGGGQ
VQLQESGPGLVKPSETLSLTCTVSGGSISSTSYYWGWIRQPPGKGLEWIGSISYSGSTYYNPSL
KSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARILWFGEYLGDYWGQGTLVTVSSASTKGPSV
TSGQA
 
SEQ ID NO:71 scFv-N038 Sfi1-fragment (NA)
NGGCCCAGGCGGCCGAGCTCCTGGTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAA
GGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAG
CAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGATTC
CTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGAC
TGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGAGTGCTTGGGTGTTCGGC
GGAGGGACCCAGCTGACCGTCCTCGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTG
GCTCGGGCGGTGGTGGAGAGGTGCAGCTGGTGCAGTCTGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCAGCAGTGGTTACTACTGGGGCTGG
ATCCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTAGCAATCATAGTGGGAGCACCT
ACTACAACCCGTCCCTCAGGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAATTCTC
CCTGAAGGTGAACTCTGTGACCGCCGCAGACACGGCCGTTTATTACTGTGCGAGAGAGGCGGGG
TATAGCAGCAGCTGGTACTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCCTCAGCCT
CCACCAAGGGCCCATCGGTCACTAGTGGCCAGGCCGGCCN
 
SEQ ID NO:72 scFv-N038 Sfi1-fragment (AA)
XAELLVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDR
FSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTQLTVLGGGSSRSSSSGGGGSG
GGGEVQLVQSGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIGSSNHSGSTYYN
PSLRSRVTISVDTSKNQFSLKVNSVTAADTAVYYCAREAGYSSSWYFDYWGQGTLVTVSSASTK
GPSVTSGQA
 
The complete people F018 of SEQ ID NO:73 γ 2 heavy chains
QLQLQESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVT
TSVDTSKNQFSLRLSSVTAADTAVYYCVAWFGDLLSLKGVELWGQGTLVTVSSASTKGPSVFPL
APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN
FGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
The complete people F018 of SEQ ID NO:74 lambda light chain
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFS
GSKSGTSASLAISGLRSEDEADYYCAAWDDSLSAWVFGGGTQLTVLGQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH
RSYSCQVTHEGSTVEKTVAPTECS
 
Complete people F063 of SEQ ID NO:75 and J042 γ 2 heavy chains
QLQLQESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVT
ISVDTSKNQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSSASTKGPSVFPL
APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN
FGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
The complete people F063 of SEQ ID NO:76 lambda light chain
QSVLTQPPSVSGTPGQRVTVSCSGSSSNIGSKNVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFS
GSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFSGGTKVTVLGQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH
RSYSCQVTHEGSTVEKTVAPTECS
 
SEQ ID NO:77: complete people J004 γ 2 heavy chains
QLQLQESGPGLVKPSETLSHTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVT
ISVDTSKNQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSSASTKGPSVFPL
APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN
FGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
The complete people J004 of SEQ ID NO:78 lambda light chain
QSVLTQPPSVSGTPGQRVTISCSGSSSNIGSSYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFS
GSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTELTVLGQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH
RSYSCQVTHEGSTVEKTVAPTECS
 
The complete people J042 of SEQ ID NO:79 lambda light chain
QSVLTQEPSVSGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGAPDRFS
GSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH
RSYSCQVTHEGSTVEKTVAPTECS
 
The complete people N049 of SEQ ID NO:80 γ 2 heavy chains
QLQLQESGPGLVKPSETLSLTCTVSGGSIWGWIRQPPGKGLEWIGSIYSSGSTYYNPSLKSRVT
ISVDTSENQFSLRLSSVTAADTAVYYCVVWFGDLLSLKGVELWGQGTLVTVSSASTKGPSVFPL
APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN
FGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
The complete people N049 of SEQ ID NO:81 lambda light chain
QSVLTQPPTVSGTPGQRVTISCSGSSSNIGSKNVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFS
GSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKVTVLGQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH
RSYSCQVTHEGSTVEKTVAPTECS
 
The complete people I022 of SEQ ID NO:82 γ 2 heavy chains
QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSYYWGWIRQPPGKGLEWIGSISYSGSTYYNPS
LKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARILWFGEYLGDYWGQGTLVTVSSASTKGPS
VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK
CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
The complete people I022 of SEQ ID NO:83 κ light chain
EIVMTQSPGTLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSG
SGSGTDFTLTISRLEPEDFAVYHCQQYYSTPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
 
The complete people N038 of SEQ ID NO:84 γ 2 heavy chains
QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGKGLEWIGSSNHSGSTYYNPSL
RSRVTISVDTSKNQFSLKVNSVTAADTAVYYCAREAGYSSSWYFDYWGQGTLVTVSSASTKGPS
VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK
CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
The complete people N038 of SEO ID NO:85 lambda light chain
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS
GSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTQLTVLGQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH
RSYSCQVTHEGSTVEKTVAPTECS
 
SEQ?ID?NO：86?HSC-F018-B
CCTGGCCGGCCTGGCCACTAGTGACCGATGGGCCCTTGGTGGAAGC

Claims (15)

1. specificity is in conjunction with the monoclonal antibody of Nicotine, and wherein said monoclonal antibody is a human monoclonal antibodies, and
(a) wherein said monoclonal antibody comprises at least one HCVR, and wherein said HCVR comprises:
(i) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1;
(ii) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2; With
(iii) HC CDR3, wherein said HC CDR3 is made up of the peptide of any in SEQ ID NO:3 and 4; Preferably, wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3, and
(b) wherein said monoclonal antibody comprises at least one LCVR, and wherein said LCVR comprises:
(i) LC CDR1, wherein said LC CDR1 is made up of the peptide of any among the SEQ ID NO:5,6 and 7, and preferably, wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5;
(ii) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8;
(iii) LC CDR3, wherein said LC CDR3 is made up of the peptide of any in SEQ ID NO:9 and 10, and preferably, wherein said LC CDR3 is made up of the peptide of SEQ ID NO:9.
2. the monoclonal antibody of claim 1, wherein
(a) the 7-117 position of described HCVR is made up of the peptide of any among the SEQ ID NO:24,28,33 and 39, and preferably, the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24; And
(b) the 5-107 position of wherein said LCVR is made up of the peptide of any among the SEQ ID NO:26,31,35,37 and 41, and preferably, the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26.
3. each monoclonal antibody in the aforementioned claim, wherein
(a) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26;
(b) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:28, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:31;
(c) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:33, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:35;
(d) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:28, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:37; Or
(e) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:39, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:41;
Wherein preferably, the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26.
4. each monoclonal antibody in the aforementioned claim, wherein said monoclonal antibody comprise at least one γ 2 heavy chain and at least one lambda light chain, wherein
(a) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:73, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:74;
(b) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:75, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:76;
(c) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:77, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:78;
(d) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:75, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:79; Or
(e) described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:80, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:81;
Wherein preferably, described γ 2 heavy chains comprise or preferably are made up of the peptide of SEQ ID NO:73, and wherein said lambda light chain comprises or preferably be made up of the peptide of SEQ ID NO:74.
5. each monoclonal antibody in the aforementioned claim, wherein said monoclonal antibody is IgG2.
6. each monoclonal antibody in the aforementioned claim, wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine.
7. each monoclonal antibody in the aforementioned claim, wherein said monoclonal antibody in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-100nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-100nM, and preferably, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-70nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-50nM, further preferably, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-10nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-10nM.
8. the HCVR of monoclonal antibody, wherein said monoclonal antibody is a human monoclonal antibodies, and wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine; The described bonded dissociation constant Kd of wherein preferably described monoclonal antibody and described (S)-(-)-Nicotine is 1-100nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-100nM; And the described bonded dissociation constant Kd of wherein further preferably described monoclonal antibody and described (S)-(-)-Nicotine is 1-70nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-50nM, further preferred 1-20nM; And further preferred again 1-10nM; And wherein said HCVR comprises:
(a) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1;
(b) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2; With
(c) HC CDR3, wherein said HC CDR3 is made up of the peptide of any in SEQ ID NO:3 and 4, and preferably wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3.
9. the HCVR of claim 8, the 7-117 position of wherein said HCVR is made up of the peptide of any among the SEQID NO:24,28,33 and 39, and wherein preferably the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24.
10. the LCVR of monoclonal antibody, wherein said monoclonal antibody is a human monoclonal antibodies, and wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine; The described bonded dissociation constant Kd of wherein preferably described monoclonal antibody and described (S)-(-)-Nicotine is 1-100nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-100nM; And the described bonded dissociation constant Kd of wherein further preferably described monoclonal antibody and described (S)-(-)-Nicotine is 1-70nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-50nM, further preferred 1-20nM; And further preferred again 1-10nM; And wherein said LCVR comprises:
(a) LC CDR1, wherein said LC CDR1 is made up of the peptide of any among the SEQ ID NO:5,6 and 7, and preferably wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5;
(b) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8;
(c) LC CDR3, wherein said LC CDR3 is made up of the peptide of any in SEQ ID NO:9 and 10, and preferably wherein said LC CDR3 is made up of the peptide of SEQ ID NO:9.
11. the LCVR of claim 10, the 5-107 position of wherein said LCVR is made up of the peptide of any among the SEQID NO:26,31,35,37 and 41, and preferably the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26.
12. monoclonal antibody, wherein said monoclonal antibody is a human monoclonal antibodies, and wherein said monoclonal antibody specificity in conjunction with (S)-(-)-Nicotine and (R)-(+)-Nicotine, the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (S)-(-)-Nicotine is 1-100nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-100nM; Wherein further preferably, the described bonded dissociation constant Kd of described monoclonal antibody and described (S)-(-)-Nicotine is 1-70nM; And the described bonded dissociation constant Kd of wherein said monoclonal antibody and described (R)-(+)-Nicotine is 1-50nM, further preferred 1-20nM; And further preferred again 1-10nM.
13. the monoclonal antibody of claim 12,
(a) wherein said monoclonal antibody comprises at least one HCVR, and wherein said HCVR comprises:
(i) HC CDR1, wherein said HC CDR1 is made up of the peptide of SEQ ID NO:1;
(ii) HC CDR2, wherein said HC CDR2 is made up of the peptide of SEQ ID NO:2; With
(iii) HC CDR3, wherein said HC CDR3 is made up of the peptide of any in SEQ ID NO:3 and 4; Preferably wherein said HC CDR3 is made up of the peptide of SEQ ID NO:3, and
(b) wherein said monoclonal antibody comprises at least one LCVR, and wherein said LCVR comprises:
(i) LC CDR1, wherein said LC CDR1 is made up of the peptide of any among the SEQ ID NO:5,6 and 7, and preferably wherein said LC CDR1 is made up of the peptide of SEQ ID NO:5;
(ii) LC CDR2, wherein said LC CDR2 is made up of the peptide of SEQ ID NO:8;
(iii) LC CDR3, wherein said LC CDR3 is made up of the peptide of any in SEQ ID NO:9 and 10, and preferably wherein said LC CDR3 is made up of the peptide of SEQ ID NO:9.
14. the monoclonal antibody of claim 12 or 13, wherein
(a) the 7-117 position of described HCVR is made up of the peptide of any among the SEQ ID NO:24,28,33 and 39, and preferably the 7-117 position of wherein said HCVR is made up of the peptide of SEQ IDNO:24; And
(b) the 5-107 position of wherein said LCVR is made up of the peptide of any among the SEQ ID NO:26,31,35,37 and 41, and preferably the 5-107 position of wherein said LCVR is made up of the peptide of SEQID NO:26.
15. each monoclonal antibody among the claim 12-14, wherein
(a) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26;
(b) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:28, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:31;
(c) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:33, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:35;
(d) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:28, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:37; Perhaps
(e) the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:39, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:41;
Wherein preferably, the 7-117 position of described HCVR is made up of the peptide of SEQ ID NO:24, and the 5-107 position of wherein said LCVR is made up of the peptide of SEQ ID NO:26.
CN2008801181882A 2007-11-29 2008-09-11 Human monoclonal nicotine specific antibodies Pending CN101878228A (en)

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